Matrices for drug delivery and methods for making and using the same

ABSTRACT

In one aspect, biocompatible matrices such as sol-gels encapsulating a reaction center may be administered to a subject for conversion of prodrugs into biologically active agents. In certain embodiments, the biocompatible matrices of the present invention are sol-gels. In one embodiment, the enzyme L-amino acid decarboxylase is encapsulated and implanted in the brain to convert L-dopa to dopamine for treatment of Parkinson&#39;s disease.

1. RELATED APPLICATION INFORMATION

[0001] This Application claims the benefit of priority under 35 U.S.C.section 119(e) to Provisional Application 60/119,828, filed Feb. 12,1999.

2. INTRODUCTION

[0002] Many clinical conditions, deficiencies, and disease states may beremedied or alleviated by providing to a patient beneficial biologicallyactive agents or removing from the patient deleterious biologicallyactive agents. In many cases, provision of beneficial agents or removalof deleterious ones may restore or compensate for the impairment or lossof function or homeostasis. An example of a disease or deficiency statewhose etiology includes loss of such an agent include Parkinson'sdisease, in which dopamine production is diminished. The impairment orloss of such agents may result in the loss of additional metabolicfunctions.

[0003] Parkinson's disease, one of many motor system disorders, resultsin symptoms such as tremor, bradykinesia, and impaired balance. Kellerin Handbook of Parkinson's Disease (Marcel-Dekker Inc.: New York 1992).Parkinson's disease is both chronic and progressive, and nearly 50,000Americans are diagnosed with Parkinson's disease each year. More thanhalf a million Americans are currently being treated for Parkinson'sdisease. Bennett et al. Dis. Mon. 38:1 (1992).

[0004] A specific area of the brain known as the basal ganglia isaffected in Parkinson's disease. The basal ganglia plays a vital role involuntary movement control. A region of the basal ganglia termed thesubstantia nigra is important in the synthesis of the neurotransmitterdopamine. Deterioration of the dopamine producing cells in thesubstantia nigra results in the characteristic symptoms of Parkinson'sdisease. These symptoms are thought to be due to a deficiency ofdopamine in both the substantia nigra and the striatum. Obeso et al.,Advances in Neurology 74:143 (1997). The striatum requires a balance ofthe neurotransmitters dopamine and acetylcholine in order to controlproperly movement, balance, and walking. The cause of the impairment ordeath of the cells responsible for the production of dopamine in thesubstantia, although currently unknown, has been attributed to a numberof factors, including oxidant stress, mitochondrial toxicity, andautoimmunity. Olanow et al., in Neurodegenetaion and Neuroprotection(Academic Press: San Diego 1996).

[0005] There are currently a number of methods being used for treatingParkinson's disease, which can be grouped into two categories, namelychemical and surgical methods. Yahr et al. Advances in Neurology 60:11-17 (1993). In chemical treatment methods, the goal is to achieve astasis between the counterbalancing dopamine and acetylcholineneurotransmitters. Jankovic et al., in Parkinson's Disease and MovementDisorders 115-568 (Williams and Wilkins: Baltimore). The correct balanceof the neurotransmitters produces a therapeutic effect in theParkinson's disease patient. At least three methods of accomplishing orrestoring a therapeutic balance are presently possible. First, in thedopaminergic method, a balance may be achieved by increasing deficientdopamine levels by using dopamine precursors or by increasing levels ofdopamine agonists in the brain. Controlled release systems have beenused to increase dopamine levels. Becker et al. Brain Res. 508:60(1990); Sabel Advances in Neurology, 53:513-18 (1990). Second, monoamineoxidase inhibitors (MAO) reduce the rate of dopamine breakdown catalyzedby monoamine oxidase enzymes and thereby increase the dopamine levels inthe brain. Third, anticholinergics block the receptor sites foracetylcholine in an attempt to compensate for low dopamine levels.

[0006] Currently, there are at least two surgical methods being utilizedin Parkinson's therapy. Jankovic et al., supra. In ablative surgeries, asmall portion of the globus pallidus (pallidotomy) or the thalamus(thalamotomy) is destroyed, which has been shown to be effective intreating Parkinson's disease. In tissue transplants, dopaminergic cells,such as fetal nigral primordia and adrenal chromaffin cells, are graftedinto the basal ganglia region or striatum. Fetal dopaminergic neuronshave been observed to provide superior functional recovery in terms ofboth magnitude and duration of effects. Kordower et al., in TherapeuticApproaches To Parkinson's Disease 443-72 (Roller et al. eds., MercerDekker Inc.: New York (1990)). This is true for both rodent and nonhumanprimate models of Parkinson's disease as well as clinical trials inParkinson's disease patients. Bakay et al. Ann. NY Acad. Sci. 495:623-40(1987); Bankiewiez et al. Progress in Brain Research 78:543-50 (1988);Freed et al. New England Journal of Medicine 327:1549-55 (1992). Inaddition, such cells have been encapsulated, Emerich et al. Neurosci.Behav. Rev. 16:437-47 (1992), and found to alleviate symptoms ofParkinson's disease in rodents, Aebischer et al. Brain Res. 560:43(1991); Lindner et al. Exp. Neurol. 132:62-76 (1995); Subramanian et al.Cell Transplant 6:469-77 (1997).

[0007] Although both chemical and surgical methods help to decrease thesymptoms of Parkinson's disease, there are a number of areas requiringimprovement. With respect to chemical methods, delivery to the striatalregion of any biologically active agent, such as dopamine, MAOinhibitors, or anticholinergics, is complicated, in part, because of thepresence of the blood-brain barrier, which may result in lowbioavailability of any such agents. As an alternative, directadministration of dopamine into the central nervous system may requirethe frequent and repeated use of invasive procedures which compromisethe integrity of the blood-brain barrier. Those techniques requirerepeated infusions into the brain, either through injections viacannulae, or from pumps which must be replaced every time the reservoiris depleted. Even with the careful use of sterile procedures, there isrisk of infection. It has been reported that even in intensive careunits, intracerebroventricular catheters used to monitor intracranialpressure become infected with bacteria after about three days. Saffran,Perspectives in Biology and Medicine 35:471-86 (1992). In addition tothe risk of infection, there seems to be some risk associated with theinfusion procedure. Infusions into the ventricles have been reported toproduce hydrocephalus, Saffran et al. Brain Research 492:245-54 (1989),and continuous infusions of solutions into the parenchyma is associatedwith necrosis.

[0008] Because of the fact that dopamine itself does not readily crossthe blood-brain barrier, many of the drug therapies utilize the dopamineprecursor L-dopa. Modern Pharmacology 108 (2d ed, Craig et al. eds,1986). Conversion of L-dopa to dopamine requires the enzyme amino aciddecarboxylase, which is found in the substantia nigra of the brain. Theprogression of Parkinson's disease and the need for larger doses ofL-dopa in order to produce therapeutic effects may be due to the loss ofthe enzyme required for this conversion. This loss of therapeuticefficacy is known as long-term L-dopa syndrome and occurs in 3 to 5years in 50% of Parkinson's disease patients being treated with L-dopa.Brannan et al. Neurology 45:596 (1991).

[0009] Surgical tissue transplantation suffers from a number of factorssuch as immunogenic complications, delayed improvement results, and lowtissue survival rates of around 10%. The use of fetal tissue hasformidable hurdles, including the failure to reestablish the normalneural circuitry, high mortality and morbidity associated with thetransplant procedure, and the ethical issue of human fetal tissueresearch. Aebischer et al. Transactions of the ASME 113:178 (1991).Adrenal cells are generally only implanted in patients less than 60years of age, as the adrenal gland of older patients may not containsufficient dopamine-secreting cells, which limits the usefulness of theprocedure as a treatment method because the disease most often affectsthe elderly. With respect to encapsulation of dopamine producing cells,questions remain concerning cell viability upon encapsulation and theirresulting durability and output. Lindner et al. Cell Transplant 7:165-74(1998).

[0010] Although the different therapies discussed above for Parkinson'sdisease have met with some success, there remains a need for additionaltreatment methods for the condition. In the present invention, in part,novel methods of producing the biologically active agent dopamine in thebrain is contemplated. In another aspect, the present inventioncontemplates treating diseases or conditions by either producing orremoving biologically active agents in a patient.

3. SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention contemplates matricesencapsulating a reaction center, and methods of using the same.

[0012] In another aspect, the present invention is directed to methodsfor producing a biologically active agent from a prodrug involvingencapsulating a cell-free reaction center in a biocompatible matrix andadministering the matrix to a subject, wherein said reaction centerconverts a prodrug into a biologically active agent in the subject. Inone method of the present invention, the matrices of the presentinvention are administered to a subject for treatment of a disease orcondition by production or removal of a biologically active agent oragents.

[0013] In another aspect, the present invention involves methods ofenzyme replacement therapy for treating a subject involvingadministering to the subject a reaction center which is encapsulated ina biocompatible matrix, wherein said reaction center replaces, augments,or supplements some activity in said subject. The reaction center may bean enzyme in which a subject to be treated is deficient, because of, forexample, a disease or condition or an inborn error of metabolism.

[0014] In another aspect, the present invention contemplates methods forthe extracorporeal use of the subject matrices in, for example, organassist devices such as a liver assist device. In one method of thepresent invention, the matrices of the present invention are used exvivo for treatment of a disease or condition by production or removal ofa biologically active agent or agents from a patient.

[0015] In certain embodiments of the present invention, including theforegoing aspects, the reaction center may be an enzyme, an antibody, acatalytic antibody or other biological material. In other embodiments,the matrix may be an inorganic-based sol-gel matrix or a silica-basedsot-gel matrix. More than one reaction center may be encapsulated in asingle matrix. In addition to any encapsulated reaction center, thematrix may have encapsulated additives. In one preferred embodiment, thereaction center may be L-amino acid decarboxylase, the prodrug may beL-dopa and the biologically active agent may be dopamine.

[0016] In still another aspect, the matrices of the present invention,and methods of using the same, may be used in diagnostic applications,such as in certain embodiments in which an imaging agent is encapsulatedtherein.

[0017] In still another aspect, the matrices and compositions of thepresent invention may be used in the manufacture of a medicament for anynumber of uses, including for example treating any disease or othertreatable condition of a patient. In still other aspects, the presentinvention is directed to a method for formulating (either separately ortogether) matrices, prodrugs and other materials and agents required fortreatment in a pharmaceutically acceptable carrier.

[0018] In another aspect, this invention contemplates a kit includingmatrices of the present invention, and optionally instructions for theiruse. For example, in one embodiment, such kits include matrices andassociated prodrug for treatment of a patient. Such kits may have avariety of uses, including, for example, imaging, diagnosis, therapy,vaccination, and other applications.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1: Enzymatic activity assay for a matrix containingβ-Glucosidase.

[0020]FIG. 2: Substrate and product spectra for penicillinase assay.

[0021] FIGS. 3(a) and (b): Penicillinase activity assays showing (a)multiple assays of a single matrix and (b) a single assay performed oneach of five matrices from one batch preparation.

[0022]FIG. 4: Change in absorbance at three hours as a function of theenzyme concentration added to the matrix during preparation.

[0023]FIG. 5: Yield of immobilized enzyme in penicillinase-containingsol-gel matrices (observed activity was calculated as the percentage ofenzyme activity used in the preparation of the matrices).

[0024]FIG. 6. Activity of crushed and whole matrices containingpenicillinase. FIGS. 6 and 7 both show data for five unique matricesassayed one time each.

[0025]FIG. 7: Penicillinase activity in whole monoliths and crushedmatrices with points shown being the mean of five measurements (errorbars +/− one standard deviation).

[0026] FIGS. 8(a) and (b): (a) Activity of penicillinase-containingmatrices with varying surface areas, and (b) activity as a percentage ofthe activity added in preparation. Surface areas corresponding to thelabeling in the graphs are: A=15.1 cm², B=39.2 cm², C=71.3 cm² and D=135 cm².

[0027]FIG. 9: Tyrosine decarboxylase activity assay; elapsed time sincegel cast 19.5 h.

[0028]FIG. 10. Tyrosine decarboxylase activity assay of two identical 16day old matrices, A and B, with comparison to C (same matrix compositionaged 19 h, no cofactor present). Assay of A is performed in the absenceof pyridoxal-5-phosphate (cofactor) while B is performed with cofactorpresent.

5. DETAILED DESCRIPTION OF THE INVENTION

[0029] 5.1. Definitions

[0030] For convenience, the meanings of certain terms employed in thespecification are provided below. The meanings for these terms, as thoseof skill in the art would understand them, should be read in light ofthe remainder of the specification for a full and complete understandingof the scope of the invention.

[0031] The term “additives” refers to compounds, materials, andcompositions that may be included in a matrix along with a reactioncenter. An additive may be encapsulated in or on a matrix or attached toa matrix, either the interior or exterior, by some interaction,including a covalent one or adhesion of the additive to the matrix.Examples of additives include other molecules necessary for theconversion mediated by the reaction center, solid materials which serveas a framework for the matrix, etc.

[0032] The term “antibody” refers to a binding agent including a wholeantibody or a binding fragment thereof which is reactive with a specificantigen. Antibodies can be fragmented using conventional techniques andthe fragments screened for utility in the same manner as described abovefor whole antibodies. For example, F(ab)2 fragments can be generated bytreating an antibody with pepsin. The resulting F(ab)2 fragment can betreated to reduce disulfide bridges to produce Fab fragments.

[0033] The term “biocompatible matrix” as used herein means that thematrix, upon implantation in a subject, does not elicit a detrimentalresponse sufficient to result in the rejection of the matrix or torender it inoperable, for example through degradation. To determinewhether any subject matrix is biocompatible, it may be necessary toconduct a toxicity analysis. Such assays are well known in the art. Onenon-limiting example of such an assay for analyzing a composition of thepresent invention would be performed with live carcinoma cells, such asGT3TKB tumor cells, in the following manner: various amounts of subjectmatrices are placed in 96-well tissue culture plates and seeded withhuman gastric carcinoma cells (GT3TKB) at 104/well density. The degradedproducts are incubated with the GT3TKB cells for 48 hours. The resultsof the assay may be plotted as % relative growth versus amount ofmatrices in the tissue-culture well. In addition, matrices of thepresent invention may also be evaluated by well-known in vivo tests,such as subcutaneous implantations in rats to confirm that they do notcause significant levels of irritation or inflammation at thesubcutaneous implantation sites.

[0034] The term “biologically active agent” as used herein means anyorganic or inorganic agent that is biologically active, e.g., producessome biological affect in a subject.

[0035] The term “encapsulated reaction center” means a reaction centerthat is contained within or on a matrix. For example, an encapsulatedreaction center may be immobilized somewhere in a silica matrix;alternatively, it may be attached to the interior or the surface of amatrix by some means other than physical confinement, such as bycovalent bonds or adhesion. Alternatively, an encapsulated reactioncenter may be located on the surface of a matrix.

[0036] The term “enzyme” refers to any polypeptide that converts aprodrug into a biologically active agent. An enzyme may be isolated fromnaturally occurring sources, or it may be prepared by recombinantmethods. An enzyme may be a fusion or chimeric protein of a polypeptidethat converts a prodrug and another polypeptide. An enzyme may be aportion or a fragment of a full-length enzyme. An enzyme may besubstantially purified, or only partially purified. Homologs, orthologs,and paralogs of an enzyme are also enzymes. For purposes of the presentinvention, an enzyme is not a catalytic antibody, a cell, or anorganism.

[0037] “Homology” refers to sequence similarity between two polypeptidesor between two nucleic acid molecules. Homology may be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. An“unrelated” or “non-homologous” sequence shares less than 40 percentidentity, though preferably less than 25 percent identity, with thesequence to which it is being compared.

[0038] The term “immunoisolatory matrix” means that the matrix uponadministration to subject minimizes the deleterious effects of thesubject's immune system on the reaction center or other contentscontained within the matrix.

[0039] The term “long-term, stable production of biologically activeagent” as used herein means the continued production of a biologicallyactive agent at a level sufficient to maintain its useful biologicalactivity for periods greater than at least about one month, morepreferably about two months, four months, six months, eight months, tenmonths, one year, one and a half years or more.

[0040] The term “matrix” means any material in which a reaction centerhas been encapsulated. For example, one type of matrix is a silica-basedsol-gel matrix. Another example of a matrix is an inorganic-basedsol-gel matrix. A matrix may have more than one type of reaction centerencapsulated.

[0041] The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0042] The phrases “parenteral administration” and “administeredparenterally” mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

[0043] A “patient” or “subject” to be treated by the present inventioncan mean either a human or non-human animal.

[0044] The phrase “pharmaceutically acceptable” is employed to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

[0045] The phrase “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a prodrug, compound,material, or composition from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical formulations.

[0046] The term “prodrug” is intended to encompass compounds, materials,and compositions which are converted by an encapsulated reaction centerinto a biologically active agent. One means of converting a prodrug to abiologically active agent is by an enzyme-catalyzed reaction. A prodrugneed not be biologically inactive itself; instead, to be a prodrug, acompound need only have some altered biological activity upon conversionby a reaction center. A prodrug may be either endogenous or exogenous toa subject. Also, many prodrugs produce more than one compounds uponcertain types of conversion, and the term “biologically active agent”when used to refer the products of such a prodrug conversion is intendedto encompass all of those products.

[0047] The term “reaction center” means any material or compound thatmay be encapsulated in a matrix and that converts or reacts a prodruginto a biologically active agent or reacts with a biologically activeagent to, for example, degrade such agent. In certain embodiments, thereaction center may be an enzyme, a catalytic antibody, or anonbiologically derived catalyst, such as those commonly used fororganic synthesis. In certain embodiments, the reaction center may beprokaryotic or eukaryotic cells, such as bacteria, yeast, or mammaliancells, including human cells, or components thereof, such as organelles.In other embodiments of the present invention, the reaction center issubstantially pure, i.e. 95%, 96%, 97%, 98% or 99% pure and thereforeessentially cell-free or organism-free. Numerous examples of reactioncenters are set forth below.

[0048] The phrases “systemic administration,” “administeredsystemically,” “peripheral administration” and “administeredperipherally” mean the administration of a compound, drug or othermaterial other than directly into the central nervous system such thatit enters the patient's system and, thus, is subject to metabolism andother like processes, for example, subcutaneous administration.

[0049] The phrase “therapeutically effective amount” means that amountof a prodrug, biologically active agent, compound, material, orcomposition according to the present invention which is effective forproducing some desired therapeutic effect. Because in certainembodiments of the present invention, a prodrug is converted into abiologically active agent by an encapsulated reaction center, it isnecessary to consider this conversion in determining what may be a“therapeutically effective amount” of a prodrug. The amount can varygreatly according to the effectiveness of a matrix, prodrug, orbiologically active agent, the age, weight, and response of theindividual subject, as well as the nature and severity of the subject'ssymptoms. Accordingly, there is no upper or lower critical limitationupon the amount of the a matrix, prodrug, or biologically active agent.The required quantity to be employed of a matrix or prodrug incombination with a matrix in the present invention may readily bedetermined by those skilled in the art.

[0050] The terms “treating” or “method of treatment” (and variationsthereof) is intended to encompass curing as well as ameliorating atleast one symptom of a condition, deficiency, or disease.

[0051] The term “ED₅₀” means the dose of a drug, including, for example,a matrix or a combination of a matrix and prodrug, which produces 50% ofa maximum response or effect. Alternatively, the dose which produces apre-determined response in 50% of test subjects or preparations.

[0052] The term “LD₅₀” means the dose of a drug, including, for example,a matrix or a combination of a matrix and prodrug, which is lethal in50% of test subjects.

[0053] The term “therapeutic index” refers to the therapeutic index of adrug, including, for example, a matrix or a combination of a matrix andprodrug, defined as LD₅₀/ED₅₀.

[0054] The term “heteroatom” as used herein means an atom of any elementother than carbon or hydrogen. Preferred heteroatoms are boron,nitrogen, oxygen, phosphorus, sulfur and selenium.

[0055] Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

[0056] The term “alkyl” refers to the radical of saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In preferredembodiments, a straight chain or branched chain alkyl has 30 or fewercarbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀for branched chain), and more preferably 20 or fewer. Likewise,preferred cycloalkyls have from 3-10 carbon atoms in their ringstructure, and more preferably have 5, 6 or 7 carbons in the ringstructure.

[0057] Moreover, the term “alkyl” (or “lower alkyl”) as used throughoutthe specification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,alkylaminos, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

[0058] The term “aralkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

[0059] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous in length and possible substitution to the alkylsdescribed above, but that contain at least one double or triple bondrespectively.

[0060] Unless the number of carbons is otherwise specified, “loweralkyl” as used herein means an alkyl group, as defined above, but havingfrom one to ten carbons, more preferably from one to six carbon atoms inits backbone structure. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

[0061] The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The term “aryl” refers to both substituted andunsubstituted aromatic rings. The aromatic ring can be substituted atone or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromaticor heteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

[0062] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

[0063] The terms ortho, meta and para apply to 1,2-, 1,3- and1,4-disubstituted benzenes, respectively. For example, the names1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

[0064] The terms “heterocyclyl” or “heterocycle” refer to 4- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, quinoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

[0065] The terms “polycyclyl” or “polycyclic group” refer to two or morerings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

[0066] The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

[0067] The phrase “fused ring ” is art recognized and refers to a cyclicmoiety which can comprise from 4 to 8 atoms in its ring structure, andcan also be substituted or unsubstituted, (e.g., cycloalkyl, acycloalkenyl, an aryl, or a heterocyclic ring) that shares a pair ofcarbon atoms with another ring. To illustrate, the fused ring system canbe a benzodiazepine, a benzoazepine, a pyrrolodiazepine, apyrroloazepine, a furanodiazepine, a furanoazepine, athiophenodiazepine, a thiophenoazepine, an imidazolodiazepine, animidazoloazepine, an oxazolodiazepine, an oxazoloazepine, athiazolodiazepine, a thiazoloazepine, a pyrazolodiazepine, apyrazoloazepine, a pyrazinodiazepine, a pyrazinoazepine, apyridinodiazepine, a pyridinoazepine, a pyrimidinodiazepine, or apyrimidinoazepine.

[0068] As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

[0069] The terms “amine” and “amino” are art-recognized and refer toboth unsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

[0070] wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen,an alkyl, an alkenyl, —(CH₂)_(m)-R₈₀, or R₉ and R₁₀ taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R₈₀ represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In preferred embodiments, only oneof R₉ or R₁₀ can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen togetherdo not form an imide. In even more preferred embodiments, R₉ and R₁₀(and optionally R′₁₀) each independently represent a hydrogen, an alkyl,an alkenyl, or —(CH₂)_(m)-R₈₀. Thus, the term “alkylamine” as usedherein means an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₉ and R₁₀is an alkyl group.

[0071] The term “acylamino” is art-recognized and refers to a moietythat can be represented by the general formula:

[0072] wherein R₉ is as defined above, and R′₁₁ represents a hydrogen,an alkyl, an alkenyl or —(CH₂)_(m)-R₈₀, where m and R₈₀ are as definedabove.

[0073] The term “amido” is art recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

[0074] wherein R₉, R₁₀ are as defined above. Preferred embodiments ofthe amide will not include imides which may be unstable.

[0075] The term “alkylthio” refers to an alkyl group, as defined above,having a sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)-R₈₀, wherein m and R₈₀ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

[0076] The term “carbonyl” is art recognized and includes such moietiesas can be represented by the general formula:

[0077] wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)-R₈₀ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)-R₈₀, where m and R₈₀ are as defined above.Where X is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R₁₁ is as definedabove, the moiety is referred to herein as a carboxyl group, andparticularly when R₁₁ is a hydrogen, the formula represents a“carboxylic acid”. Where X is an oxygen, and R′₁₁ is hydrogen, theformula represents a “formate”. In general, where the oxygen atom of theabove formula is replaced by sulfur, the formula represents a“thiolcarbonyl” group. Where X is a sulfur and R₁₁ or R′₁₁ is nothydrogen, the formula represents a “thiolester.” Where X is a sulfur andR₁₁ is hydrogen, the formula represents a “thiolcarboxylic acid.” WhereX is a sulfur and R′₁₁ is hydrogen, the formula represents a“thiolformate.” On the other hand, where X is a bond, and R₁₁ is nothydrogen, the above formula represents a “ketone” group. Where X is abond, and R₁₁ is hydrogen, the above formula represents an “aldehyde”group.

[0078] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)-R₈₀, where m and R₈₀ are described above.

[0079] The terms “sulfoxido”, as used herein, refers to a moiety thatcan be represented by the general formula:

[0080] in which R′₁₁ is as defined above, but is not hydrogen.

[0081] A “sulfone”, as used herein, refers to a moiety that can berepresented by the general formula:

[0082] in which R′₁₁ is as defined above, but is

[0083] not hydrogen.

[0084] The term “sulfonamido” is art recognized and includes a moietythat can be represented by the general formula:

[0085] in which R₉ and R′₁₁ are as defined above.

[0086] The term “sulfamoyl” is art-recognized and includes a moiety thatcan be represented by the general formula:

[0087] in which R₉ and R₁₀ are as defined above.

[0088] A “phosphoryl” can in general be represented by the formula:

[0089] wherein Q₁ represented S or O, and R₄₆ represents hydrogen, alower alkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

[0090] wherein Q₁ represented S or O, and each R₄₆ independentlyrepresents hydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N.When Q₁ is an S, the phosphoryl moiety is a “phosphorothioate”.

[0091] A “phosphoramidate” can be represented in the general formula:

[0092] wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, Sor N.

[0093] A “phosphonamidate” can be represented in the general formula:

[0094] wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S.

[0095] Analogous substitutions can be made to alkenyl and alkynyl groupsto produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

[0096] The definition of each expression, e.g. alkyl, m, n, etc., whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

[0097] Certain compounds of the present invention may exist inparticular geometric or stereoisomeric forms. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

[0098] If, for instance, a particular enantiomer of a compound of thepresent invention is desired, it may be prepared by asymmetricsynthesis, or by derivitization with a chiral auxiliary, where theresulting diastereomeric mixture is separated and the auxiliary groupcleaved to provide the pure desired enantiomers. Alternatively, wherethe molecule contains a basic functional group, such as amino, or anacidic functional group, such as carboxyl, diastereomeric salts areformed with an appropriate optically-active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers.

[0099] Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the desired use ofthe compound.

[0100] It will be understood that “substitution” or “substituted with”includes the implicit proviso that such substitution is in accordancewith permitted valence of the substituted atom and the substituent, andthat the substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, hydrolysis, etc.

[0101] As used herein, the term “substituted” is contemplated to includeall permissible substituents of organic compounds. In a broad aspect,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

[0102] For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 67th Ed., 1986-87, insidecover. Also for purposes of this invention, the term “hydrocarbon” iscontemplated to include all permissible compounds having at least onehydrogen and one carbon atom. In a broad aspect, the permissiblehydrocarbons include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic organic compoundswhich can be substituted or unsubstituted.

[0103] By the terms “amino acid residue” and “peptide residue” is meantan amino acid or peptide molecule without the —OH of its carboxyl group(C-terminally linked) or the proton of its amino group (N-terminallylinked). In general the abbreviations used herein for designating theamino acids and the protective groups are based on recommendations ofthe IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry11:1726-1732 (1972)). For instance Met, Ile, Leu, Ala and Gly represent“residues” of methionine, isoleucine, leucine, alanine and glycine,respectively. By the residue is meant a radical derived from thecorresponding α-amino acid by eliminating the OH portion of the carboxylgroup and the H portion of the α-amino group. The term “amino acid sidechain” is that part of an amino acid exclusive of the —CH(NH₂)COOHportion, as defined by Kopple, Peptides and Amino Acids 2, 33 W. A.Benjamin Inc., New York and Amsterdam, 1966; examples of such sidechains of the common amino acids are —CH₂CH₂SCH₃ (the side chain ofmethionine), —CH(CH₃)—CH₂CH₃ (the side chain of isoleucine),—CH₂CH(CH₃)₂ (the side chain of leucine) or H— (the side chain ofglycine).

[0104] For the most part, the amino acids used in the application ofthis invention are those naturally occurring amino acids found inproteins, or the naturally occurring anabolic or catabolic products ofsuch amino acids which contain amino and carboxyl groups. Particularlysuitable amino acid side chains include side chains selected from thoseof the following amino acids: glycine, alanine, valine, cysteine,leucine, isoleucine, serine, threonine, methionine, glutamic acid,aspartic acid, glutamine, asparagine, lysine, arginine, proline,histidine, phenylalanine, tyrosine, and tryptophan. However, the termamino acid residue further includes analogs, derivatives and congenersof any specific amino acid referred to herein. For example, the presentinvention contemplates the use of amino acid analogs wherein a sidechain is lengthened or shortened while still providing a carboxyl, aminoor other reactive precursor functional group for cyclization, as well asamino acid analogs having variant side chains with appropriatefunctional groups. For instance, such amino acid analogs includeβ-cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine,homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan,1-methylhistidine, or 3-methylhistidine. Other naturally occurring aminoacid metabolites or precursors having side chains which are suitableherein will be recognized by those skilled in the art and are includedin the scope of the present invention.

[0105] Also included are the D and L stereoisomers of such amino acidswhen the structure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols D, L or DL, furthermore when theconfiguration is not designated the amino acid or residue can have theconfiguration D, L or DL. It will be noted that the structure of some ofthe compounds of this invention includes asymmetric carbon atoms. It isto be understood accordingly that the isomers arising from suchasymmetry are included within the scope of this invention. Such isomersare obtained in substantially pure form by classical separationtechniques and by sterically controlled synthesis. For the purposes ofthe present invention, unless expressly noted to the contrary, a namedamino acid shall be construed to include both the D or L stereoisomers,preferably the L stereoisomer.

[0106] The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed. (Greene et al., ProtectiveGroups in Organic Synthesis, 2^(nd) ed.; Wiley: New York, 1991).

[0107] The phrase “N-terminal protecting group” or “amino-protectinggroup” as used herein refers to various amino-protecting groups whichcan be employed to protect the N-terminus of an amino acid or peptideagainst undesirable reactions during synthetic procedures. Examples ofsuitable groups include acyl protecting groups such as, to illustrate,formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl andmethoxysuccinyl; aromatic urethane protecting groups as, for example,carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups suchas t-butyloxycarbonyl (Boc) or 9-Fluorenylmethoxycarbonyl (FMOC).

[0108] The phrase “C-terminal protecting group” or “carboxyl-protectinggroup” as used herein refers to those groups intended to protect acarboxylic acid group, such as the C-terminus of an amino acid orpeptide. Benzyl or other suitable esters or ethers are illustrative ofC-terminal protecting groups known in the art.

[0109] 5.2. Uses

[0110] As a general introduction to the present invention, one way toappreciate certain aspects of the invention is by considering some ofthe different uses to which certain embodiments may be put. Therendition of these uses is for illustrative purposes only, and suchcategorization is not intended to limit the scope of the presentinvention. Other uses may be readily apparent to those of skill in theart, and other features of the present invention are presented below andmay be applicable to any of the following uses. For all these uses,certain embodiments of the present invention may be used in animalmodels for assaying new treatments, e.g., new therapeutics or newtreatment regimes.

[0111] 5.2.1. Prodrug Activation

[0112] In one aspect of the present invention, embodiments of thepresent invention may be used as prodrug activators, that is, forprodrug activation. When used in this fashion, the reaction centerencapsulated in a matrix reacts with a prodrug or prodrugs to produce abiologically active agent or agents. The prodrug may be exogenous to thesubject, thereby requiring administration of the prodrug, or the prodrugmay be endogenous to the subject, in which case administration of theprodrug to a subject may be used to add to the prodrug present in thesubject, but is not absolutely necessary. For prodrug activation, onefeature concerns matching the prodrug of interest with the reactioncenter encapsulated so that the reaction center may convert the prodruginto a biologically active agent. In general, the therapeutic effect ofa matrix used for pending activation may vary greatly with its site ofadministration.

[0113] One related example of prodrug activation using exogenous sourcesinvolves ADEPT technology, or antibody directed enzymatic prodrugtherapy, whereby an enzyme that converts a prodrug into a cytotoxicagent is attached to an antibody that is targeted to a antigen ofinterest, often a surface cell receptor of a neoplastic growth. Seegenerally Denny et al. J. Pharm. Pharmacol. 50:387-94 (1998) and U.S.Pat. Nos. 6,015,556, 6,005,002, and 5,985,281. After binding of theantibody-enzyme conjugate to the antigen, prodrug is administered. Theenzyme converts the exogenous prodrug into the cytotoxic agent in thevicinity of the neoplastic growth. In this fashion, ADEPT technologyreduces toxicity to the subject because the cytotoxic agent is onlyproduced in the immediate vicinity of the tumor. In gene-directed enzymeprodrug therapy (GDEPT), the exogenous enzyme is generated selectivelyin the tumor cells after delivery of a DNA construct containing thecorresponding gene. The present invention contemplates relying onbystander effects and the like in the same fashion by administration,e.g., implantation, of the matrix in the vicinity of any neoplasm,whereupon administration of a prodrug causes conversion of that prodruginto a biologically active agent adjacent to the neoplasm. In contrastto ADEPT, the matrix may not be cleared from the subject as is oftenobserved for the antibody-enzyme conjugate, so multiple administrationsof prodrug using the present invention may be feasible. The presentinvention may also not result in nonspecific activation of the prodrug,which may occur using ADEPT if the antibody-enzyme conjugated bindsindiscriminately or is not cleared by treatment with another antibodyprior to administration of the prodrug.

[0114] Another illustrative report of prodrug activation using aexogenous source involved localized generation of 5-flurouracil from5-fluorocytosine by surgically implanting immobilized cytosine deaminaseadjacent to subcutaneous tumors in rats and injecting intraperitomeally5-fluorocytosine. Nishiyama et al., Cancer Res. 45:1753-61 (1985). Inthis example, the implanted enzyme was encapsulated in a dialysis tube.

[0115] Embodiments of the present invention that may be grouped underthis category involve the production of dopamine by a matrix, to whichan example described below is directed. The biosynthesis of dopamineinvolves a number of enzymatic steps. One traditional treatment ofParkinson's disease uses an immediate precursor of dopamine, L dopa.Although L dopa therapy is effective in reducing Parkinson's symptoms,there is a lose of L dopa efficacy over time. One possible means ofovercoming such a loss of efficacy involves increasing the enzymaticactivity necessary to convert L dopa to dopamine. One enzyme that may beused in this regard is aromatic L-amino acid decarboxylase (AADC, E.C.4.1.1.28). Cells stably expressing AADC have been grafted into6-hydroxy-dopamine denervated rat striatum, and upon administration of Ldopa, the dopamine content was observed to increase. Kaddis et al. J.Neurochem. 68:1520-26 (1997).

[0116] AADC catalzyes the irreversible decarboxylation reaction ofseveral aromatic L-amino acids, including L-dopa, m-tyrosine,p-tyrosine, phenylalanine, 5-hydroxytrptophan, and tryptophan. Hayashiet al. Biochemistry 32:812-18 (1993); Dominici et al. Eur. J. Biochem.169:209-13 (1987); Voltattorni et al. Methods in Enzymology, 142:179-87(1987); Sourkes, Methods in Enzymology, 142:170-87 (1987); Lindstrom,Biochem Biophys. Acta 884:276-81 (1986); Jung Bioorganic Chem. 14:429-43(1986); Nishigaki Biochem. J. 252:331-35 (1988). AADC is a pyridoxalphosphate dependent enzyme and is produced in the substantia nigralcells. Nigrastriatal cell death and concomitant decrease in AADCactivity may result in decreased dopamine levels in the brain.Encapsulation of AADC as the reaction center and appropriateadministration may allow for production of dopamine from L-dopa in thenigrastriatal region for treatment of Parkinson's disease.

[0117] It is possible that, by using this embodiment or related ones ofthe present invention, it may not be necessary to administer L-dopa toproduce a therapeutic effect. Encapsulated enzymes may not requireexogenous prodrug, e.g., administration of L-dopa, because L-dopa levelsthat occur naturally in a subject may be a sufficient source of dopamineupon conversion by the matrix. As a result, side effects of L-dopaadministration common in present therapeutic treatments, includingnausea, may be avoided. L-dopa therapy presently requires simultaneousadministration of a peripheral decarboxylase inhibitor, such ascarbidopa or benserazide, to reduce decarboxylation of L-dopa todopamine outside the brain, which causes such nausea. Calne et al. NewEng. J. Med. 329:1021-27 (1993).

[0118] Although AADC is the enzyme responsible for L-dopa conversion todopamine within the substantia nigra region of the brain, at least oneadditional enzyme is capable of effecting this conversion. L-Tyrosinedecarboxylase (TD, E.C. 4.1.1.25) catalyzes the removal of the carboxylgroup from tyrosine to produce tyramine and carbon dioxide. Pyridoxal5′-phosphate is a necessary coenzyme. Although TD has greaterspecificity for decarboxylation of L-tyrosine to tyramine, TD alsocatalyzes decarboxylation of L-dopa. Maraques et al. Plant Physiol, 88:46-51 (1988). Accordingly, TD may be encapsulated as the reaction centerin a matrix of the present invention as a treatment method forParkinson's disease.

[0119] In another approach to Parkinson's treatment using an embodimentof the present invention, an encapsulated reaction center may be used toproduce a precursor of dopamine, such as L-dopa. Such a matrix would besimilar to traditional L-dopa treatments, but in this case, L-dopa wouldbe produced by the matrix only in the location where the matrix wasadministered. In this fashion, the matrix could be implanted so thatL-dopa would be produced only in the brain. Consequently, there wouldprobably be no side effects as reported for traditional L-dopa therapy.Tyrosine is converted to L-dopa by the enzyme tyrosine monooxygenase(TMO, E.C. 1.14.16.2). Encapsulation of TMO in a matrix andadministration to the brain should increase L-dopa levels there.Tyrosine readily crosses the blood-brain barrier and would result invery little systematic toxicity. Tyrosine may or may not be necessary toadminister to a subject to achieve the desired therapeutic effect.

[0120] In another embodiment of the present invention, it is possible toencapsulate both TMO and either AADC, TD, or both, in a matrix. By doingso, it would be possible for a single matrix to convert tyrosine toL-dopa, and L-dopa to dopamine. Because the decarboxylating enzyme,either AADC or TD, would be in close proximity to any L-dopa produced bythe TMO, by virtue of both enzyme being encapsulated in the same matrix,conversion to dopamine readily proceed. Because tyrosine is less toxicthan L-dopa, it may be desirable to use a matrix to effect twoconversions, instead of just one. The present invention contemplatesencapsulating more than one reaction center per matrix. Yamanka et al.J. Sol-Gel Sci. & Tech. 7:117-21 (1996).

[0121] In another embodiment, the enzyme monophenol monooxygenase (MMOE.C. 1.14.18.1), or tyosinase, is encapsulated as the reaction center.MMO is the key enzyme in melanin synthesis, catalyzing the first twosteps of the pathway: dehydroxylation of L-tyrosine to L-dopa andoxidation of L-dopa to dopaquinone. The former reaction is termedcresolase activity, and the later reaction is termed catecholaseactivity. A number of assays have been used to measure the tyrosinasehydroxylase and dopa oxidase activities, including spectrophotometric,radiometric, HPLC and electrometric methods. Winder, J. Biochem.Biophys. Methods 28:173-83 (1994); Vachtenheum et al., AnalyticalBiochem. 146:405-10 (1985). MMOs from a number of different sources areknown. Kenji Adachi et al. Biochem. Biophys. Res. Comm. 26 (1967);Seymour H. Pomerantz, Tyrosinases (Hampster Melanoma) 620-626; Duckworthet al., J. Biol. Chem. 245:1613-25 (1970); Steiner et al., AnalyticalBiochem, 238:72-75 (1996). Oxidation of o-diphenols to benzoquinones isreferred to as catecholase activity. Although catecholase activity ofMMO may reduce the production of the desired therapeutic L-dopa product,engineering of the sol-gel matrix may allow for increased production ofthe diphenol product. For example, it has been reported thatadministration of liposome-entrapped tyrosinase to rat increases levelsof L-dopa in rat plasma. Miranda et al. Gen. Pharmacol. 24:1319-22(1993). In the present invention, if the matrix is administered in thebrain, then the increase in L-dopa would occur where it would have thegreatest therapeutic effect.

[0122] In another aspect of the present invention, modulation ofdopamine and related neurotransmitters may have use in treatment forcocaine addiction. See U.S. Pat. No. 5,189,064. Chronic cocaine usersmay experience dopamine deficiency, and dopamine supplementation likethat contemplated by the present invention may reduce the feeling ofdysphoria inadequate stimulation attributable to depressed dopaminelevels, which invites readministration of the drug or recividism.

[0123] In another aspect, the present invention contemplates applyingthe matrix-based technology to modulate the availability of anycompounds by augmenting the enzymes found in the biological pathway forany such compounds. For example, tryptophan is converted into5-hydroxy-tryptophan by the enzyme tryptophan hydroxylase withconcomitant conversion of tertrahydrobiopterin to dihydrobiopterin. AADCthen converts 5-hydroxy-tryptophan to serotonin, which is aneurotransmitter. Serotonin is found in the gastrointestinal tract andin the brain, where it is synthesized locally in the pineal gland.Monoamine oxidase converts serotonin into 5-hydroxy-indoleacetaldehyde,which aldehyde dehydrogeanse metabolizes into 5-hydroxy-indoleaceticacid. The present invention contemplate encapsulation any one or more ofthe enzymes in the serotonin pathway either to produce, as was discussedfor the dopamine example discussed above, or to degrade serotonin invivo as clinically necessary to treat any disease or condition. Thus,those of skill in the art may be able to use the present invention tomodulate any biological pathway using enzymes of such pathway asreaction centers.

[0124] Some other possible enzymes, which may be useful inneuropharmacology and which may be used as reaction centers in thepresent invention, and the reaction that they catalyze, are listedbelow: Enzyme Chief Reactant Chief Product Choline acetyltransferaseacetyl CoA + choline CoA + o-acetylcholine Phenylalanine 4-L-phenylalanine L-tyrosine monooxygenase Dopamine β- dopaminenorepinephrine monooxygenase (noradenaline) Noradrenalin N- noradrenalinadrenaline methyltransferase (epinephrine) Monoamine oxidasenorepinephrine 3,4-dihydroxy- phenylglycolaldehydeCatecholamine-O-methyl norepinephrine 3-O- transferase (COMT)methylnorepinephrine Histidine decarboxylase histidine histamineHistamine histamine 1-methylhistamine methyltransferase Diamine oxidasehistamine 5-imidazole acetic acid Diamine oxidase 1-methylhistamine1-methylimidazole acetic acid L-Glutamic acid-1- glutamateγ-aminobutyric acid decarboxylase (GABA) GABA-α-oxoglutarateγ-aminobutyric glutamate and succinic transaminase acid (GABA) (plus α-semialdehyde Serine hydroxymethylase oxoglutarate) L-serine glycine

[0125] See generally Enzymes (Dixon et al. eds; 3d ed. 1979).

[0126] In addition to these illustrative examples, the present inventioncontemplates providing any biologically active agents to treat a diseaseor condition. For example, in the nervous system, chronic, low-leveldelivery of trophic factors is sufficient to maintain the health ofgrowth-factor dependent cell populations. In chronic disorders such asAlzheimer's disease and Huntington's disease, long-term delivery of oneor more neurotrophic factors such as NGF, BDNF, NT-3, NT-4/5, CNTF, GDNFand CDF/LIF may be required to maintain neuronal viability. These growthfactors cannot be delivered through systemic administration as they areunable to traverse the blood-brain barrier. Therefore, it may benecessary to deliver such neurotrophic factors into the central nervoussystem as a prodrug that is able to cross the blood-brain barrier. Thepresent invention contemplates preparing prodrugs of such biologicallyactive agents, and using an encapsulated reaction center to convert theprodrug into such an agent in the central nervous system. For example,CNTF is a potential therapeutic agent for Huntington's disease. Emerichet al. Nature 386:395-99 (1997). As discussed in more detail below, oneof skill in the art could prepare a prodrug for CNTF so that a specificencapsulated reaction center would be capable of converting the prodrugto CNTF. As one example of such a matching between a prodrug and areaction center, insulin may be obtained from recombinant proinsulin byreaction with carboxypeptidase or trypsin. Markvicheva et al. App.Biochem. Biotechnol. 61:75-84 (1996).

[0127] In certain embodiments of the present invention, an antibody maybe encapsulated as the reaction center and the matrix administered tothe subject. The matrix would be used to isolate deleteriousbiologically active agents from the subject in the matrix, as opposed tomodifying as would a reaction center that reacts with such abiologically active agent. After all the antibodies have bound theircorresponding hapten, the matrix may or may not be removed from thesubject.

[0128] In certain embodiments matrices of the present invention may beused as prodrug activators in vivo after administration of the matrixand, if necessary, any prodrug, to a subject. Alternatively, the presentinvention contemplates using a matrix to convert a prodrug into abiologically active agent ex vivo, whereupon the biologically activeagent is administered to a subject, as described in more detail in U.S.Pat. No. 5,378,232.

[0129] 5.2.2. Enzyme Replacement, Augmentation, or Supplementation

[0130] In other aspects of the present invention, a reaction center isencapsulated in a matrix to replace or augment some lost biologicalactivity that is generally present in a healthy subject, e.g., arewithout the disease or condition to be treated. Certain embodiments ofthe production of dopamine described above are examples of suchreplacement or augmentation. The encapsulated reaction center mayrestore or augment vital metabolic functions, such as the removal oftoxins or harmful metabolites. Lost biological activity might resultfrom loss of activity generally, or loss of activity in some location,e.g., a specific type of tissue. The loss may be attributable to agenetic defect, for example, an inborn error of metabolism, or may becaused by some disease or condition. If loss of function is complete,then this aspect of the invention may be referred to as enzymereplacement, whereas if loss of function is only partial, then thisaspect of the invention may be referred to as enzyme augmentation. Inother embodiments, although there has been no loss of enzyme function,enzyme augmentation may produce a desirable therapeutic effect. In otherembodiments, the enzyme encapsulated is new to the subject, that is,there is supplementation. Through use of certain embodiments of thepresent invention, homeostasis of particular substances can be restoredand maintained for extended periods of time.

[0131] Based on conditions and diseases that those of skill in the artknow to result from loss of a particular enzymatic activity, reactioncenters that replace or augment lost or diminished biological activitymay be readily identified. Certain embodiments of the present inventionhave advantages over more conventional types of enzyme replacementtherapy (ERT), which often rely on administration of an enzyme whoseactivity is lost or diminished. The matrices of the present inventionfor the most part may be biocompatible and immunoisolatory with respectto any encapsulated reaction center, whereas enzyme administration canresult in hypersensitivity and/or anaphylactic reaction during orimmediately after enzyme infusion. Brooks et al., Biochem. Biophys. Acta1497:163-72 (1998). The development of antibodies to any enzyme used inERT may preclude its use in long-term therapeutic regimes or followingrelapses. Likewise, the present invention may avoid the need todermitize an enzyme used in ERT with polyethylene glycol (PEG). Goldberget al. Biomedical Polymers 441-52 (Academic Press 1980). Certainembodiments of the present invention, by encapsulating an enzyme as thereaction center, prevent degradation, and thereby may provide forprolonged treatment upon administration of the matrix. In contrast, forERT, multiple infusions of the enzyme may be required for sustainedtherapy. Even erythrocyte-entrapped enzymes may show only modestincreases in activity. See, for example, Thorpe et al. Pediatr. Res.9:918-23 (1975).

[0132] By way of illustration, ERT has been used to treat Gaucher'sdisease. See generally Morales, Ann. Pharmacother. 30:381-88 (1996).Gaucher's disease is caused by a genetic deficiency of the enzymeglucocerebrosidase, and results in accumulation of glucocerebrosidasewithin the reticuloedothelial system. Symptoms includehepatosplenomegaly, bone marrow suppression, and bone lesions. There arethree subtypes, of which the most common, type 1, is non-neuronopathic.Types 2 and 3, which are neuronopathic, may result in nerve celldestruction. Enzyme treatment first began with a placentally derivedform of glucocerebrosidase, Dale et al. Proc. Natl. Acad. Sci. USA73:4672-74 (1976), which has been replaced with a recombinant version ofthe enzyme. The enzyme treatment must be repeated every two weeks, andit has been effective in reducing hepatosplenomegaly, improving anemiaand thrombocytopenia, and general health. Numerous studies have beencompleted on ERT for Gaucher's disease. Magnaldi et al., Eur. Radiol.7:486-91 (1997); Ueda et al., Acta Paediatr. Jpn. 38:260-04 (1996);Lorberboym et al., J. Nucl. Med. 38:890-95 (1997); Charrow et al., ArchInter. Med. 158:1754-60 (1998). The present invention contemplatesencapsulation of this enzyme in a matrix and administration to a subjectsuffering from Gaucher's disease for treatment.

[0133] ERT has also been effected by using materials that act by slowrelease. For example, L-asparaginase has been loaded into nanoparticlescomposed of polymers that release the enzyme over a few weeks, or loadedinto erythrocytes, Updike et al. J. Lab. Clin. Med. 101:679-91 (1983).The enzyme may be useful for treating cancer, especially acutelymphocytic leukemia. By encapsulating this enzyme as the reactioncenter in a matrix, the enzymatic activity may be present for longerperiods than that made possible by using slow-release methods.

[0134] In another embodiment of the present invention, transportproteins such as hemoglobin may be encapsulated to produce an artificialred blood cell. In such an embodiment, the matrix must be of appropriatemorphology to travel throughout the vasculature of a subject.

[0135] In addition to the examples already discussed, almost anynaturally occurring enzyme may be used in to augment or replaceenzymatic activity, and is therefore a candidate for this use. Someother possible enzymes that may be used as reaction centers, and thedisease or condition that they may treat, are listed below. (Theseenzymes may be used to treat other diseases and conditions as well.)Enzyme Disease or condition Reference (Pompe's disease) α-GalactosidaseFabry's disease (heart and kidney failure due to ceramide accumulation)α-L-iduronidase mucopolysaccharidosis Kakkis et al. Biochem. Mol. typeI. Med. 58: 156-67 (1996) β-glucuronidase mucopolysaccharidosis typeO'Connor et al. J. Clin. VII Invest. 101: 1394-400 (1998)Aminolaevulinate Lead poisoning Bustos et al. Drug Des. Deliv.dehydratase 5: 125-31 (1989) Bilirubin oxidase jaundice CatalaseAcatalasemia Fibrinolysin Thromboembolic occlusive vascular diseaseGlutaminase (e.g., from Cancer Pseudomonas putrefaciens) HemoglobinRespiratory Heparinase (e.g., from Extracorporeal circulationFlavobacterium heparinum) L-arginine ureahydrolase HyperargininemiaWissmann et al. Somot. Cell (A1), Arginase Mol. Genet. 22: 489-98 (1996)Liver microsomal enzymes Liver failure Brunner et al. Artif. Organs(e.g., from rabbit liver) 3: 27-30 (1979); U.S. Pat. No. 5,849,588Phenylalanine ammonia lyase Phenylketonuria Bourget et al. Biochim(e.g., from Rhodotorula Biophys Acta 883: 432-48 glutinis) (1986)Streptokinase (e.g., from Thromboembolic occlusive Streptococcus sp.)vascular disease Superoxide dismutase (e.g., Inflammatory diseasesLedwozyw Acta Vet Hung from bovine liver), catalase thought to bemediated by 39: 215-24 (1991); Turrnes et oxygen free radicals, e.g.,al. J. Clin. Invest. 73: 87-95 bleomycin-induced lung (1984) fibrosisTerrilythin Peritonitis Tyrosinase Liver failure UDP Glucuronyltransferase Jaundice, liver disease (e.g., from rabbit liver) Urea cycleenzymes Liver failure Urease Renal failure Uricase (e.g., from hogliver) Hyperuricemia due to gout Urokinase (e.g., from humanThromboembolic occlusive urine) vascular disease

[0136] 5.2.3. Addiction Neutralization

[0137] In another aspect of the invention, the encapsulated reactioncenter is chosen to degrade biologically active agents that may resultin addiction. Efforts to combat addiction, e.g., cocaine addiction, haveincluded inducing anti-drug antibodies specific to the drug. Seegenerally U.S. Pat. No. 5,840,307. In certain embodiments, the presentinvention may help to neutralize the addiction, or in other words, causeaddiction neutralization. For example, investigators have encapsulatedalcohol dehydrogenase and/or acetaldehyde dehydrogeanse in humanerthrocytes and reported the continuous degradation of ethanol for up toseventy hours. Lizano et al. Biochem. Biophys. Acta 1425:328-36 (1998).See also U.S. Pat. No. 5,759,539. Encapsulation of either or both ofthese enzymes in a matrix may allow for the complete metabolization ofethanol upon administration of the matrix, which would thereby combataddiction by neutralizing the addictive agent, ethanol.

[0138] In another example, catalytic antibodies have been elicited thatare capable of aiding hydrolysis of the cocaine molecule. Landry et al.Science 259:1899-1901 (1993). See also U.S. Pat. No. 5,730,985.Encapsulation of such catalytic antibodies in a matrix of the presentinvention and administration to a subject addicted to cocain may allowfor neutralization of any ingested cocaine. The present invention, byencapsulating the catalytic antibody, may avoid some of the drawbacksusually associated with passive antibody therapy. In the same fashion,reaction centers targeted at biologically active agents responsible forother types of addition are known to those of skill in the art, and maybe used in a similar fashion.

[0139] 5.2.4. Mutagenic Assays

[0140] In another aspect of the invention, the present inventioncontemplates using matrices as metabolic activating systems for use, forexample, as toxicology screens for cytotoxic and pharmaceuticalcompounds in vivo. Such a use may reduce the need for laboratory animalsfor toxicology testing. Numerous efforts have been made to prepare humanliver epithelian cell lines, and liver cell and tissue culture systemsfor such uses. See, for example, U.S. Pat. Nos. 5,849,588 and 5,759,765.In one report, enzymes responsible for deactivation of many endogenoustoxins have been isolated and covalently bound onto a hemocompatibleform of agarose support. Brunner et al., Artif. Organs 3:27-30 (1979).In a like manner, the present invention contemplates encapsulating suchenzymes in matrices of the present invention and evaluating suspectedmutagens. See generally Rueff et al Mutat. Res. 353:151-76 (1996).Enzymes usually found in the liver, such as cytochrome P-450 forexample, may be used in this or related embodiments. Janig et al. ActaBiol. Med. Ger. 38:409-22 (1979).

[0141] 5.2.5. Tissue Assist Devices

[0142] In addition to the many methods and uses described herein inwhich the subject matrices are administered for in vivo use, thematrices of the present invention may be used ex vivo. Many of theteachings herein described for in vivo use apply as well to ex vivo use(and visa-versa).

[0143] In one aspect of the present invention, one example of an ex vivouse is a tissue assist device, and in certain embodiments, an organassist device. In such a device, matrices encapsulating one or morereaction centers could be used to replace, augment or supplement thebiological function of an organ or other tissue. It is important to notethat for this embodiment (and others described herein in which theprodrug converted by the reaction center is potentially deleterious tothe subject being treated), it is not always necessary that the prodrugbe converted into the same agent(s) that the enzymes and other catalystsusually present in the tissue would have were it to react with theprodrug. In certain embodiments of the subject invention, it is not theproduct of the prodrug that is critical; instead it is thetransformation of the prodrug into a less toxic or otherwise undesirablecompound(s) that is the primary concern. For example, this principleapplies to certain of the embodiments used for addition neutralizationdescribed above as well as certain tissue assist devices.

[0144] One example of such an assist device is particularly well-suitedto the subject matrices is an hepatic assist device. Livertransplantation has become widely accepted as an effective treatment forchronic and acute liver disease. One of the major problems associatedwith the transplantation process, however, as been the need for aneffective means for providing temporary support for patients awaiting anavailable donor organ. Extracorporeal devices that are effective forliver support has proven more elusive. See, for example, Takahashi etal., Digestive Diseases and Sciences 36(9) (1991).

[0145] In certain embodiments of the present invention, one or moreenzymes generally localized in the liver of a patient could beencapsulated in a subject matrix, and blood and other bodily fluids ofthe patient could be passed through and over these matrices ex vivo toaugment the biological activity usually associated with the liver.Functions of the liver that could be addressed by such liver assistdevices include, among others, carbohydrate, fat and protein metabolismand detoxification of drugs, hormones and other substances. At the pointthat the encapsulated reaction centers of the matrices are exhausted orotherwise less efficient than desired, they may be readily replaced byproviding with new and more efficient matrices.

[0146] A variety of reaction centers could be encapsulated for such aliver assist device. Examples include cytochrome P-450, other enzymesusually located in the liver, a less than highly purified mixture ofbiologicals isolated from livers, transformed cells such as thosederived from hepatoblastoma cell lines (Sussman et al., Hepatology16:60-65 (1992)), cultured or isolated hepatocytes (U.S. Pat. No.5,866,420; Rozga et al., Hepatology 17:258-65 (1993); Rozga et al., Ann.Surg. 217:502-11 (1994)), cells from hepatocarcinoma-derived cell lines(Richardson et al., J. Cell Biol. 40:236-47 (1969); Aden et al., Nature(London) 282:615-16 (1970)), Kupffer cells and other biologicals thatare capable of replacing, augmenting or supplementing the biologicalfunction of the liver. For other examples of possible biologicals andliver assist devices, see, for example, U.S. Pat. Nos. 6,008,049,5,849,588, 5,290,684, 5270,192, 5,043,260, 4,853,324, 3,734,851; and WO93/16171. Sources of suitable enzymes and biologicals for a liversassist device for humans include, for example, porcine and othermammals. In particular, use of certain biologicals, including forexample hepatocytes, has proved difficult because their instability, andencapsulation of such biologicals in matrices of the present inventionmay improve their stability.

[0147] A variety of bioreactor techniques known to those of skill in theare could be used with such an assist device, including for example,hollow fiber techniques, static maintenance reactor systems, fluidizedbed reactors, microporous membranes and flat-bed, single-pass perfusionsystems. See, for example, U.S. Pat. Nos. 4,200,689, 5,081,035,3,997,396; and WO 90/13639; Halberstadt et al., Biotechnology andBioengineering 43:740-46 (1994).

[0148] In addition to liver assist devices, other organs or functions ofa patient could be treated using matrices of the present invention exvivo.

[0149] 5.3. Matrices

[0150] The concept of encapsulating or immobilizing a reaction center inor on a matrix of some kind is well precedented. For example,significant efforts have been made to immobilize enzymes on solidsupports. Handbook of Enzyme Biotechnology (2d ed., ed. Wiseman 1985).In these and other examples, encapsulation or immobilization of thereaction center may impart desirable characteristics on the reactioncenter.

[0151] A number of matrix chemistries that may be used in the presentinvention have been used to immobilize enzymes or biologically activeagents. For instance, cells have been attached to glass beads andimplanted in rats. Cherksey et al. Neuroscience 75:657-64 (1996).Reaction centers may be immobilized on a type of porous zirconia. Huckelet al., J. Biochem. Biophys Methods 31:165-79 (1996). Alternatively,reaction centers may be attached to supports through silane coupling.Weetall, Appl. Biochem. Biotechnol. 41:157-88 (1993). Biologics may beimmobilized within a composite fibre by using a gel formation ofcellulose derivative and metal alkoxide, e.g., titanium isopropoxide.Hatayama et al. J. Sol-Gel Sci. & Tech. 7:13-17 (1996); Ohmori et al. J.Biotechnol. 33:205-09 (1994). Poly(vinyl alcohol) synthetic polymerfoams may be used. Li et al. J. Biomater. Sci. Polm. Ed. 9:239-58(1998). Other polymers known in the art may be used. See, for example,U.S. Pat. Nos. 5,529,914 and 5,780,260; WO 93/16687. As described ingreater detail below, inorganic-based or silica-based sol-gel matricesare contemplated by the present invention. Some examples of suitableinorganic-based matrices include those disclosed in the followingreferences: Mazei et al., J. Materials Chemistry 8:2095-101 (1998);Yoldas, J. Mater. Sci. 1098-92 (1986); and Curran et al., Chemistry ofMaterials, 10:3156-66 (1998).

[0152] One feature of the matrix in certain embodiments of the presentinvention is its ability to prevent leaching of any encapsulatedreaction center, at least to the extent necessary for the intended useof the matrix. In certain embodiments of the present invention, theremay be negligible leaching. In others, there may be some leaching, butusually only a small amount over time. If leeching proses to beexcessive, either the material to be encapsulated, e.g., a reactioncenter or an additive, or the sol-gel matrix may be modified to improveleaching characteristics, e.g., reduce leaching. For example, to reduceleaching, the reaction center may be derivatized to increase its size.For example, an enzyme may be chemically modified to create derivativesby forming covalent or aggregate conjugates with other chemicalmoieties, such as glycosyl groups, lipids, phosphate, acetyl groups,PEG, and the like. Covalent derivatives may be prepared by linking thechemical moieties to functional groups on amino acid sidechains of theprotein or at the N-terminus or at the C-terminus of the polypeptide.Alternatively, a fusion or chimeric polypeptide retaining at least someof the activity of the enzyme may be used. Alternatively, the reactioncenter of additive may be attached to the sol-gel matrix in somefashion, e.g., by covalently bonding.

[0153] The manner in which a reaction center is encapsulated in amatrix, be it for example by physical entrapment, covalent attachment,or some other physical attraction, may affect the properties of suchreaction center. For example, the micro environment around anycovalently attached reaction center may differ from that encountered bythe same reaction center encapsulated during gelation of the sol-gel,and any difference may affect the activity of the center. Thus, thepresent invention contemplates adjusting encapsulation, if necessary,for each intended use.

[0154] In preparing any matrix, the encapsulated material, e.g. thereaction center and additives, must be robust enough to retain theirusefulness after being encapsulated. For example, many biologicalmaterials may not be able to survive the high temperatures and harshconditions required to prepare some inorganic materials. Consequently,such inorganic materials may not be used with sensitive biolgicals. Inthe present invention, matrices are matched with the reaction center(s)or additive(s) to be encapsulated therein so as to retain sufficientactivity of the reaction center.

[0155] Another feature of the present invention is the ability of thematrix to stabilize, in certain cases, the encapsulated reaction center.For example, the present invention may protect against degradation ofany encapsulated biological material by naturally occurring systems,such as proteases. The matrix may protect against thermal denaturationof any encapsulated biological materials. Finally, the matrix may evenassist in the correct re-folding of any denature polypeptide chain.Heichal-Segal et al. Bio/Technology 13:798 (1995).

[0156] 5.3.1. Silica-Based Sol-Gel Matrices

[0157] In one aspect of the present invention, reactions centers areencapsulated in silica-based sol-gel matrices. Silica-based sol-gelshave been applied to encapsulate a wide range of materials, includingbiological materials, small organic molecules, antibodies, antigens, andorganic catalysts. See, for example, Ellerby et al. Science 255:1113-15(1992); Dave et al. Analytical Chem. 66:1120-27 (1994); Avnir et al.Acc. Chem. Res., 28:328-34 (1995); Avnir et al. Chem. Mater., 6:1605-14(1994) (listing encapsulated purified enzymes and whole-cell extractsand whole cells); Biochemical Aspects of Sol-Gel Science and Technology(eds. Avnir et al. 1996); Shtelzer et al. Biotechnol. Appl. Biochem.15:227-35 (1992). Biological materials encapsulated in inorganic-basedor silica-based sol-gel matrices have retained significant activity fora substantial time period.

[0158] Inorganic-based sol-gels, and in particular, silica-basedsol-gels, have a variety of characteristics that are useful forencapsulation of reaction centers and implantation in vivo. Seegenerally Dunn et al. Acta Mater. 46:737-41 (1998); Avnir et al. Chem.Mater., 6:1605-14 (1994). Any or all of these features may or may not bepresent in particular embodiments of the present invention. Some suchfeatures include: stability to heat, light (no photodegredation), andelectrical current (no electrochemical degradation); transparent in thevisible region and into the UV-Vis region; controllable surface area andporosity (average pore size and pore size distribution); possibility ofcontrolling conductivity by appropriate use of other inorganic alkoxidesduring preparation of the gel or addition of additives; capable of beingreadily modified chemically; improved stability of any encapsulatedmaterial, e.g., reaction center or additive, because of the rigidmatrix; little or no leaching of any encapsulated material; readilymanipulated in a variety of physical morphologies; and isolatory of anyencapsulated material from the surrounding environment, except for anysubstance that is able to diffuse into the matrix. Many of thesefeatures are explained in greater detail below.

[0159] One area of interest involves using doped sol-gels as chemicalsensors. As part of that effort, sol-gels have been used to encapsulateenzymes and antibodies. Avnir, Acc. Chem. Res., 28: 328-334 (1995);Akbarian et al. J. Sol-Gel Sci & Tech. 8:1067-70 (1997). Imuunosensorshave been prepared using sol-gel technology. Wang et al. Anal. Chem.15:1171-75 (1998). For example, the enzyme glucose oxidase has beenexamined upon encapsulation in a silica-based sol-gel matrix for use asa glucose sensing material. Yamanaka et al. Chem. Mater. 4:495 (1992);Audebert et al. Chem Mater. 5:911-13 (1993). Such sol-gel preparationshave been used as electrodes for electrochemical assays of glucoseconcentrations. Sampath et al. J. Sol-Gel Sci. & Tech. 7:123-28 (1996).

[0160] Another area of interest of silica-based sol-gel technology hasbeen encapsulating enzymes for use as organic catalysts in a variety ofapplications, including synthesis of chiral materials. Jaeger et al.TIBTECH 16:396-403 (1998).

[0161] Silica-based sol-gel matrices have also been used ascontrolled-release carriers of biologically active agents. See, forexample, U.S. Pat. No. 5,849,331 and WO 97/45367.

[0162] (a) Preparation

[0163] Modifications in well-known sol-gel processes permit theincorporation of enzymes or other biologically derived reaction centersin silica-based sol-gel matrices. See generally Avnir et al. Chem Mater.6:1605 (1994); U.S. Pat. Nos. 5,824,526, 5,650,311; 5,650,311;5,371,018; 5,308,495; 5,300,564; 5,292,801.

[0164] Silica-based sol-gel matrices of the present invention may beprepared in the sol-gel method by polymerization of a metal alkoxideprecursor. See generally Bruce Dunn et al. Chem. Mater., 9:2280-91(1997). The polymerization process is well documented and known toproceed by the formation of colloidal silica particles. A suspension ofthese particles is termed a sol. The synthesis generally involves theuse of metal alkoxides which may undergo hydrolysis and condensationpolymerization reactions. The preparation process can ordinarily bedivided into the following steps: forming a solution, gelation, ageing,drying, and densification. In the preparation of a silica-based matrix,one starts with an appropriate alkoxide, for example, Si(OC₂H₅)₄,tetraethyl orthosilicate or TEOS, or Si(OCH₃)₄, tetramethylorthosilicate or TMOS, which is mixed with water and a solvent, e.g.,the alcohol of the alkoxide, ethanol or methanol, to form a solution. Anumber of reactions result, including hydrolysis, which leads to theformation of silanol groups Si—OH, and condensation, which givessiloxane Si—O—Si groups.

[0165] There are several parameters which influence the hydrolysis andcondensation polymerization reactions, including the temperature,solution pH, particular alkoxide precursor and solvent, and relativeconcentrations of the alkoxide precursor, water, and solvent. Suchparameters may be important to retaining activity when the reactioncenter encapsulated is an enzyme or other biological. For example, inencapsulating enzymes, greater enzyme activity may been preserved by notadding any alcohol at the start of polyermization. Another improvementmay result from buffering the reaction solution to some pH suitable forany pH-sensitive materials to be encapsulated after the acid-catalyzedhydrolysis of the oxysilanes. Ellerby et al. Science 255:1113-15 (1992);Rietti-Shati et al. J. Sol-Gel Sci. & Tech. 7:77-79 (1996).

[0166] Initial hydrolysis of the precursor alkoxide is catalyzed byprotons or hydroxide ions. It is possible to control the matrixcharacteristics by controlling the rates of the individual steps bywhich the matrix is condensed. Acidic catalysis tends to increase therate of hydrolysis and disfavors the condensation reactions necessary toform the sol-gel, whereas base hydrolysis produces rapid condensation.If the reaction center to be encapsulated is not sensitive to pHconditions, the formation of the gel matrix can be achieved fairlyrapidly. However, in the case of reaction centers or additives which maybe sensitive to extreme pH conditions, such as enzymes, the pH of thesol must be adjusted prior to addition. Hence, preparation of thesilica-based sol-gel may involve buffering the sol before the additionof the reaction center or other additives. For example, to retain theactivity of bacteriorhodopsin, the solution was buffered to pH 9 afteraddition of the polypeptide. Weetall et al. Biochem Biophys. Acta1142:211-13 (1993).

[0167] As the hydrolysis and condensation of polymerization reactionscontinue, viscosity increases until the solution ceases to flow. Thissol-gel transition is irreversible, and at this stage the one-phaseliquid is transformed to a two-phase system. The gel may consist ofamorphous primary particles of variable size (5-10 nm or smaller) withan interstitial liquid phase. At this stage the pores have yet to shrinkand the liquid phase fills the pores. After gelation, gels are generallysubjected to an aging process during which the gels are sealed and verylittle solvent loss or shrinkage occurs. Condensation reactionscontinue, increasing the degree of cross-linking in the network.

[0168] The drying process involves the removal of the liquid phase.Ambient temperature evaporation may be employed, and there isconsiderable weight loss and shrinkage. It is at this stage that porecollapse may occur, deceasing pore size and thus decreasing the solventvolume. The combination of these effects causes an increase in theinteraction between the reaction center and the matrix. The final stageof the sol-gel process is that of densification. It is at this pointthat the gel-to-glass conversion occurs and the gel achieves theproperties of the glass. Matrices are found to contract to one eighththe pre-dried volume and are termed “xero-gels.” The drying process mayaffect the accessibility of any encapsulated reaction center, and byadjusting such process, the present invention contemplates another meansof influencing the activity of any encapsulated reaction center.Wamboldt et al. J. Sol-Gel Sci & Tech 7:53-57 (1996).

[0169] (b) Composition and Characteristics

[0170] Any number of alkoxide precursors may be used in preparingsilica-based sol-gel matrices of the present invention. Thosesilica-based sol-gel matrices prepared from oxysilanes other thanSi(OR¹)₄ are known as organically modified silica matrices, or Ormosilmatrices. In preparing the matrices of the present invention, forexample, alkoxides of the form Si(OR¹)₄, R²Si(OR¹)₃, R² ₂Si(OR¹)₂, R²₃Si(OR¹) may be used, in which each R¹ is independently methyl, ethyl,or any lower-weight alkyl (although the identity of R¹ is usually thesame in any type of oxysilane), and R² is independently any alkyl, aryl,or other substituent that does not interfere substantially withformation of the sol-gel, as discussed in more detail below. Asignificant difference between R¹ and R² is that the R¹ alkoxide themajority of R¹ is hydrolyzed during gelation, whereas the R² substituentremains part of the matrix. Because R² is not hydrolized but remains inthe sol-gel matrix, the identity of R² may have a significant affect onthe sol-gel matrix and any material encapsulated therein. In contrast,for R¹, much of which is hydrolized during gelation, may not constitutea significant percentage of the sol-gel matrix that results. Even so,the identity of R¹ may be important to the reaction, because HOR¹, whichis produced upon hydrolysis of the oxysilane, may affect the formationof the sol-gel and any material encapsulated therein. Accordingly, forexample, some biolgicals to be encapsulated may be stable to some HOR¹and not others. In certain preferred embodiments, the alkoxide used isSi(OR¹)₄, in which R¹ is methyl or ethyl.

[0171] In certain embodiments, R² may contain functional groups. Forexample, aminopropyl, which has an amine functional group, andmercaptopropyl, which has a thiol functional group, have been used as R²in preparing sol-gel matrices from R²Si(OR¹)₃ and mixtures of R²Si(OR¹)₃and Si(OR¹)₄, Collino et al. J. Sol-Gel Sci. & Tech 7:81-85 (1996);Venton et al. Biochim Biophys Acta 1250:117-25 (1995). Such sol-gelmatrices were used to prepare thin films. Almost any chemical moiety maybe used as R² in the present invention as long as any functional groupscontained therein are not adverse to formation of the sol-gel matrix.For those functional groups that may not be compatible with the sol-gelchemistry, it may be possible to protect them using standard protectiontechnologies know in the art of organic chemistry and then deprotectthem after preparation of the sol-gel matrix is complete.

[0172] Functional groups may be used to increase the stability orreactivity of any encapsulated reaction center, especially when thereaction center is a biologic. For example, functional groups havinghydrolizable functional groups, such a phenol or amine, may affect thelocal pH, thereby improving reactivity or stability of the encapsulatedreaction center, or directly assist catalysis or stabilize an enzyme.Alternatively, functional groups may affect the characteristics of thesurface of the matrix, which may affect the biocompatability of thematrix. In addition, by incorporating functional groups into thematrices of the present invention, the exterior characteristics of thematrix may be altered by derivitization. For example, chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups, PEG, and thelike, could be attached to the surface of the matrix though suchfunctional groups.

[0173] In other embodiments of the present invention, R² may incorporatechemistry that allows for covalent attachment of any reaction center oradditive directly to the sol-gel matrix. For example, a reaction centeror additive could be covalently attached to the silicon of an oxysilaneas R² through a linker bound to the silicon or other inorganic. Such amodified silica alkoxide could be reacted with nonsubstituted silicaalkoxides to form the sol-gel of interest. In another embodiment, themoiety covalently attached to the silica alkoxide could be a biotingroup, and the reaction center or additive could be attached to avidin.The biotin/avidin interaction would effectively attach the reactioncenter or additive to the silica oxide framework of any sol-gel matrix.An antibodyapten pair could be used in the same fashion.

[0174] For certain embodiments, the alkoxide used may be a single silicaalkoxide, or a mixture of silica alkoxides. Reetz et al. J. Sol-Gel Sci.& Tech. 7:35-42 (1996). When using silica oxides of structureR²Si(OR¹)₃, R² ₂Si(OR¹)₂, R² ₃Si(OR¹), it may be necessary to react themwith sufficient Si(OR¹)₄ to allow for adequate gelation and formation ofa physically robust matrix. For instance, the oxysilanes substitutedwith non-hydrolizable substituents may constitute 0.1, 1, 2, 5, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the oxysilane usedin the preparation. If a oxysilane has non-hydrolizable substituents,the percent of that oxysilane may need to be less to ensure sufficientgelation.

[0175] Modification of the framework of a silica-based sol-gel byvariation of the precursor alkoxide presents the possibility oftailoring the microenvironment of the encapsulated reaction center. Inthis fashion, it is possible to, for example, maximize the reactivity ofany encapsulated reaction center. Increasing the number of alkyl groupsas well as increasing chain length of the alkyl groups in the precursormaterial may produce increased matrix hydrophobicity. Suchhydrophobicity, for example, may be conducive to stabilization of thereaction center. The local pH within the sol-gel may also be affected bythe chemical identity of the matrix. In one report, the activity of anencapsulated enzyme increased upon using a mixture of oxysilanes withvarious alkyl substituents. Reetz et al. Angew. Chem. Int. Ed. Engl.34:301-303 (1995). By varying the chemical identity and ratio ofdifferent oxysilanes, the present invention contemplates customizing thesol-gel matrix for each encapsulated reaction center, e.g., to maximizecatalytic activity. Other features of the matrix may depend on thechemical identity of the oxysilane precursors, for example wettability,which may affect the biocompatibility of any matrix upon implantation.For example, cell adhesion and growth may depend on the wettability ofthe matrix. Altankov et al. J. Biomed. Mater. Res. 30:385-91 (1996);Altankov et al. J. Biomater. Sci. Polym. Ed. 8:299-310 (1996).

[0176] Pore size is an important characteristic of any sol-gel matrix,because it may affect what materials, e.g., prodrugs, may diffuse in andout of the matrix, and the leachability of any encapsulated reactioncenter(s) and/or additive(s). A number of reports indicate that the poresize and/or shape may be varied by adjusting the synthetic conditions bywhich any material is encapsulated in a sol-gel. Dave et al. ACS Symp.Ser. 622:351 (1996). The present invention contemplates pore sizesranging from the angstrom level to the micron level.

[0177] In addition to preparing silica-based sol-gel matrices fromsilica oxides, other oxides, including metal oxides, may be used toencapsulate a reaction center in inorganic-based sol-gel matrices. Inone report, glucose oxidase was encapsulated within vanadium pentaoxide,and the resulting sol-gel was used in electrochemical studies. Glezer etal. J. Am. Chem. Soc., 115:2533-34 (1993). A vanadium alkoxide has beenco-condensed with TEOS, thereby imparting the properties, includingreactivity, of oxovanadium(V) functional groups to the matrix. Stiegmanet al. Chem. Mater. 5:1591-94 (1993). Oxysilines and other metal oxidesmay be combined in any sol-gel matrix. Silica-based sol-gel matrices inwhich redox active metal ions constitute part of the sol-gel frameworkmay prove useful in promoting reactions involving electron transferssuch as reductions and oxidations within the sol-gel itself. Forexample, an electron source foreign to the matrix may transfer anelectron to a reaction center encapsulated in such a mixed metal sol-gelby electron transfer through a pathway involving the metal atoms in theframework of the sol-gel. Soghomonian et al. Chem. Mater. 5:1595-97(1993).

[0178] The local environment, or micro-environment, immediatelysurrounding any encapsulated reaction center of the present inventionmay play an important role in affecting the capability of such reactioncenter to catalyze the conversion of prodrug to biologically activeagent. A number of studies have been completed to better characterizethe nature of the microenvironment around any encapsulated material in asol-gel. Samuel et al. Chem Mater., 6:1457-61 (1994); Zheng et al. AnalChem. 69:3940-49 (1997); Dave et al. J. Sol-Gl Sci & Tech. 8:629-34(1997); Avnir et al. J. Phys. Chem. 88:5956-59 (1984). By adjusting anyof the variables delimited above, the properties of the silica-basedsol-gel matrix may be readily tailored by one of skill in the art to thereaction center encapsulated.

[0179] 5.3.2. Other Features of the Matrix

[0180] The matrix may be immunoisolatory with respect to the reactioncenter or other contents. Use of immunoisolatory matrices allows theimplantation of alkogenetic or xenogeneic reaction centers and otheradditives, without a concomitant need to immunosuppress the subject.Using immunoisolatory matrices, it is possible to implant reactionscenters that are foreign to the subject, such as nonmamallian enzymes,provided that critical substances necessary to the mediation ofimmunological attack are excluded from the implant. These substances maycomprise the complement attack complex component Clq, or they maycomprise phagocytic or cytotoxic cells; the instant immunoisolatorymatrix protects against these harmful substances.

[0181] The present invention allows for coating or otherwise modifyingthe exterior of the matrix. Such a coating may render the matriximmunoisolatory. U.S. Pat. No. 5,676,943. For example, the coating orother modification may confer protection of the reaction center or othercontents from the immune system of the host in whom the matrix isimplanted, by providing a physical barrier sufficient to preventdetrimental immunological contact between the reaction center and otheradditives and the host's immune system. The thickness of a coating mayvary, but it will always be sufficiently thick to prevent direct contactbetween the reaction center and the elements of the host's immunesystem. The thickness generally ranges between 5 and 200 microns;thicknesses of 10 to 100 microns are preferred, and thickness of 20 to75 microns are particularly preferred. Types of immunological attackwhich can be prevented or minimized by the use of a coating or othermodification include attack by macrophages, neutrophils, cellular immuneresponses (e.g. natural killer cells and antibody-dependent Tcell-mediated cytoloysis (ADCC), and humoral response (e.g.,antibody-dependent complement mediated cytolysis).

[0182] Various polymers and polymer blends can be used to manufacture acoating, including polyacrylates (including acrylic copolymers),polyvinylidenes, polyvinyl chloride copolymers, polyurethanes,polystyrenes, polyamides, cellulose acetates, cellulose nitrates,polysulfones, polyphosphazenes, polyacrylonitriles,poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymersand mixtures thereof.

[0183] The solvents used in conjunction with the above-identifiedpolymers in forming the coating will depend upon the particular polymerchosen. Suitable solvents include a wide variety of organic solventssuch as alcohols and ketones generally as well as dimethylsulfoxide(DMSO), dimethylacetamide (DMA), and dimethylformamide (DMF) and blendsof these solvents as well.

[0184] The coating may also include a hydrophobic matrix such as anethylene vinyl acetate copolymer, or a hydrophilic matrix such as ahydrogel. The coating may be post-production coated or treated with animpermeable outer layer such as a polyurethane, ethylene vinyl acetate,silicon, or alginate.

[0185] The polymeric solution for the coating may also include variousadditives such as surfactants to enhance the formation of porouschannels and antioxidants to sequester oxides that are formed during thecoagulation process. Exemplary surfactants include Triton-X 100available from Sigma Chemical Corp. and Pluronics P65, P32, and P18.Exemplary anti-oxidants include vitamin C (ascorbic acid) and vitamin E.

[0186] In addition, anti-inflammatory agents can also be incorporatedinto the coating to reduce immune response. Exemplary anti-inflammatoryagents include corticoids such as cortisone and ACTH, dexamethasone,cortisol, interleukin-1 and its receptors and agonists, and antibodiesto TGF, interleukin-1, or gamma-interferon. Alternatively, thesematerials may be added to the implant after formation by a post-coatingor spraying process. For example, the implant could be immersed in asolution containing an anti-inflammatory agent.

[0187] Post-coating procedures can also be used to provide a protectivebarrier against immunogens and the like. For example, after formation,the matrix may be coated (e.g., by immersion, spraying or applying aflowing fluid, if applicable) with a surface protecting material such aspolyethylene oxide or polypropylene oxide to inhibit proteininteractions of the reaction centers of the matrix with entities of thesubject. Other protective coatings include silicon and hydrogels such asalginates. Derivatives of these coating materials, such as polyethyleneoxide-polydimethyl siloxane, may also be used.

[0188] The coating may be formed freely around the core without chemicalbonding, or alternatively, the coating may be directly cross-linked tothe material of the implant.

[0189] The present invention also allows for enclosing the matrix in amembrane. The membrane may allow for passage of substances up to apredetermined size but prevents the passage of larger substances. Morespecifically, the surrounding or peripheral region is produced in such amanner that it has pores or voids of a predetermined range of size. As aresult, the vehicle is selectively permeable. The molecular weightcutoff (MWCO) selected for a particular coating will be determined inpart by the use contemplated. Membranes useful in the instant inventionare ultrafiltration and microfiltration membranes.

[0190] If necessary, the present invention also allows for modificationor additions to be made to the matrix to support or strengthen thematrix. U.S. Pat. No. 5,786,216. For example, structural materials, suchas a hollow tube or cylindrica support, may be encapsulated in thematrix to improve its compression strength, tensile strength, or otherproperties.

[0191] The present invention also allows for attachment of a tether tothe matrix to make implantation, placement, or recovery of the matrixmore facile. The present allow also contemplates altering the surface ofthe matrix, for example by encapsulating a structure that affects thesurface of the matrix, as an aid in fixing the matrix upon implantation.

[0192] 5.3.3. Additives

[0193] Additives may be encapsulated in the matrix in addition to anyreaction center or centers. Such additives may be used to alter theproperties of the matrix. Investigations show that the addition of lowconcentrations of organic molecules to sol-gels has very little effecton the network formation of the sol-gel. Dunn et al. Chem. Mater9:2280-91 (1997).

[0194] For example, additives that may be used in the present inventioninclude sodium fluoride and polyethylene glycol. The use of polyethyleneglycol in place of any alcohol during condensation of the sol-gel matrixmay improve enzymatic activity of the encapsulated enzyme. Likewise,sodium fluoride may be used and may improve enzymatic activity of theencapsulated enzyme. Avnir et al. Chem. Mater. 6:1605-14 (1994).

[0195] For example, additives may alter the porosity of the matrix.Alternatively, additives may be used to provide necessary reactants forany encapsulated reaction center. For example, coenzymes for a reactioncenter may be encapsulated along with the enzyme itself. For AADC andTD, a required cofactor is pyridoxal phosphate. Pyridoxal phosphate maybe encapsulated directly during preparation of the sol-gel matrix.Alternatively, pyridoxal phosphate itself may be prepared in a slowrelease formulation, and the formulation encapsulated. See generallyU.S. Pat. No. 5,759,582.

[0196] Alternatively, additives may improve the physical characteristicsof the matrix, for example, for purposes of handling and administrationof the material. As with the reaction center, additives may bephysically encapsulated during the synthesis of the sol-gel, or they maybe covalently attached to the matrix directly.

[0197] Another possible additive of the present invention allows forready detection of the matrix after administration. In this fashion, thematrices of the present invention may also be used for diagnosticpurposes. Thus an X-ray contrast agent, such as a poly-iodo aromaticcompound, may be encapsulated in the matrix. Matrices according to theinvention may also contain paramagnetic, superparamagnetic orferromagnetic substances which are of use in magnetic resonance imaging(MRI) diagnostics. Thus, submicron particles of iron or a magnetic ironoxide may be encapsulated into the matrix to provide ferromagnetic orsuper paramagnetic particles. Alternatively, paramagnetic MRI contrastagents, which principally comprise paramagnetic metal ions, such asgadolinium ions, ligated by a chelating agent which prevents theirrelease (and thus substantially eliminates their toxicity), may beencapsulated. Matrices of the present invention may also containultrasound contrast agents such as heavy materials, e.g. barium sulphateor iodinated compounds such as the X-ray contrast agents referred toabove, to provide ultrasound contrast media.

[0198] 5.3.4. Matrix Morphology

[0199] The matrices of the present invention may take any variety ofmorphologies. As a practical matter, the form of the matrix mayinitially be determined by the vessel in which the matrix issynthesized. Such matrices may subsequently be processed in order toproduce matrices of desired morphology. The size of any matrix maydepend on its intended use, and the present invention contemplatespreparing matrices having dimensions of centimeters (1, 10, 100, 1000cm), millimeters (1, 10, 100 mm), micrometers (1, 10, 100), nanometers(1, 10, 100), and picometers (0.01, 0.01, 1, 10, 100 pm). Alternatively,the size of a matrix may be referred to by mass, which the presentinvention contemplates may range anywhere from 1000 gm to 1 ng.

[0200] The matrix may be any configuration appropriate for providingsufficient activity of the encapsulated reaction center necessary forits intended use. Possible morphologies include cylindrical,rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical.For use in a subject, a matrix of the present invention may provide, inat least one dimension, sufficiently close proximity of any reactioncenters to the surrounding tissues of the subject, including thesubject's bloodstream, in order to make any biologically active agentproduced by the reaction center bioavailable.

[0201] If the matrix is to be retrieved after it is implanted,configurations which tend to prevent migration of the matrix from thesite of implantation may be desirable; in contrast, if the matrix isintended to migrate throughout a patient, other morphologies, such asspherical capsules small enough to travel in the recipient's bloodvessels, may be desirable. The degree of miniaturization of any matrixmay affect mobility and localization of a matrix in a subject. Certainshapes, such as rectangles, disks, or cylinders may offer greaterstructural integrity.

[0202] The surface area of the matrix may be important for its use. Agreater surface area may result in a greater observed activity withrespect to any particular load of an encapsulated reaction center. Inparticular, small bead or sphere shaped materials ranging in size ofradius from may be desirable because they have increased surface area ascompared to other morphologies. In order to increase further the surfacearea of a matrix, a powder of the matrix may be desirable. It may bedesirable to enclose a matrix, especially if the matrix is in powderform, in a capsule. See, for example, U.S. Pat. Nos. 5,653,975;5,773,286.

[0203] It may be possible to control the size of any matrix by using theaqueous core of reverse cellular micellar droplets as host reactors forpreparation of the matrix, as reported by Jain et al. J. Am. Chem. Soc.120:11092-95 (1998) for a silica-based sol-gel matrix. The matrixparticles prepared in such a fashion may be highly monodispersed andhave a narrow size distribution. Other hollow spheres may be used toprepare matrices of similar dimensions. See for example Caruso et al.Science 282:1111-13 (1998), U.S. Pat. No. 5,770,416, Lu et al., Nature398:223-26 (1999).

[0204] 5.4. Reaction Centers

[0205] A wide variety of compounds or materials may be used as thereaction center in the present invention. In general, any compound ormaterial that converts a compound into a biologically active agent maybe encapsulated. Alternatively, in other embodiments, any compound ormaterial that degrades a biologically active agent may be encapsulated.Alternatively, any compound or material exhibiting reactivity ofinterest may be encapsulated. Many possible reaction centers havealready been described in setting forth some of the uses of the presentinvention.

[0206] Possible types of reaction centers contemplated by the presentinvention include, for example, enzymes, catalytic antibodies,antibodies, and non-biologically derived catalysts. Many reactioncenters may be biologically derived. Numerous reports describeencapsulating enzymes in sol-gels, and such teachings may be ofassistance in embodiments of the present inventions. Zink et al. New J.Chem., 18:1109-15 (1994); Miller et al. J. Non-Crystalline Solids.202:279-89 (1996); Ji et al. J. Am. Chem. Soc., 720: 222-23 (1998);Braun et al. J. Non-Crystalline Solids 147&148:739-43 (1992); Yamanakaet al. Chem. Mater. 4:497-500 (1992); Lin et al. J. Sol-Gel Sci. & Tech.7:19-26 (1996). Catalytic antibodies have been encapsulated in sol-gelmatrices and the resulting matrices used in either a batch-wiseoperation or in a continuous flow apparatus for preparative scaleorganic synthesis. Shabat et al. Chem. Mater. 9:2258-60 (1997).Optically active polypeptides have been encapsulated with retention ofactivity. For example, bacteriorhodopsin or mutated forms have beenencapsulated in sol-gel matrices, which are optically transparent.Weetall et al. Biochem Biophys. Acta 1142:211-13 (1993); Wu et al. Chem.Mater. 5:115-20 (1993). Phycobiliproteins have also been encapsulated.Chen et al. J. Sol-Gel Sci. & Tech. 7:99-108 (1996). Antibodies againstsmall organic antigens have been encapsulated within the sol-gel.Bronshtein et al. Chem. Mater. 9:2632-39 (1997); Turniansky et al. J.Sol-Gel Sci. & Tech. 7:135-43 (1996). Wang et al. Chem. Mater.65:2671-75 (1993). Alternatively, antigens have been encapsulated. Rouxet al. J. Sol-Gel Sci. & Tech. 8:663-66 (1997); Livage et al. J. Sol-GelSci. & Tech. 7:45-51 (1996). Biologics having novel magnetic properties,such as ferritin have been encapsulated. Lan et al. J. Sol-Gel Sci. &tech. 7:109-116 (1996). In certain embodiments, the reaction centerencapsulated need not be substantially purified from its natural source.Bresslar et al. J. Sol-Gel Sci. & Tech. 7:129-33 (1996).

[0207] Cells and organisms have been encapsulated in silica-basedsol-gel matrices. Peterson el al. P.S.E.B.M. 218:365-69 (1998). Bacteriahave been immobilized in sol-gel matrices to metabolize herbicides forenvironmental clean-ups. Rietti-Shati et al. J. Sol-Gel Sci. & Tech.7:77-79 (1996). In using cells in a subject, it may be important toaccustom them to the implantation site before implantation to improvetheir viability. Differences in conditions such as glucoseconcentration, oxygen availability, nutrient concentrations, in vitroand in vivo may have an adverse affect on implantation of cells. Seegenerally U.S. Pat. Nos. 5,550,050; 5,620,883.

[0208] In addition to biological reaction centers, a variety of othermaterials have been encapsulated in silica-based sol-gels. For example,organic fluorescent dyes and photochromic information recordingmaterials have been encapsulated. Avnir et al. J. Phys. Chem. 88:5956-59(1984); Avnir et al. Journal of Non-Crystalline Solids, 74:395-406(1985); Levy et al., Journal of Non-Crystalline Solids, 113:137-45(1989). It is also possible to entrap a reaction center in a sol-gel foruse in organic catalysis. For example, lipases may be encapsulated in asol-gel for use as a heterogeneous biocatalyst. Reetz et al. Angew.Chem. Int. Ed. Engl. 34:301-03 (1995).

[0209] For those embodiments of the present invention that involveadministration of a sol-gel matrix to a subject, the reaction centerencapsulated therein need not be native to the subject. The sol-gelmatrix, as discussed above, may be immunoisolatory itself or modified tomake it so.

[0210] Any number of different types of reaction centers may beencapsulated in a single matrix. By encapsulating more than type ofreaction center in a single matrix, certain embodiments of the presentinvention may cause the conversion of one compound into a second andthen into a third, and so on. Yamanka et al. J. Sol-Gel Sci. & Tech.7:117-21 (1996); Chang et al. Artif. Organs 3:38-41 (1979). Such aresult may be advantageous if, for example, the biological activity ofthe second compound is undesirable. Alternatively, it may be the casethat it is the third compound that has a more valuable biologicalactivity than the second. Encapsulating more than one reaction centermay increase the activity of the second reaction center in any pathway.For instance, the local concentration of reactants for the second centermay be increased because of the reactivity of the first center. Fosselet al. Eur. J. Biochem. 30:165-71 (1987). Alternatively, for example, ifthe first type of reaction center catalyzes an oxidation or reduction,the second type of reaction center could mediate electron transfer andthereby facilitate greater catalytic activity. For instance, theelectron-transfer redox pair Cc:CcP complex has been encapsulated. Linet al. J. Sol-Gel Sci. & Tech. 7:19-26 (1996).

[0211] In addition to reaction of the reaction center with a singleother compound, e.g., a prodrug, the present invention also contemplatesthe reaction of more than one material with the encapsulated reactioncenter. For example, it has been shown that NADP (nicotinamide adeninedinucleotide phosphate ester) may react with the reaction center andanother molecule. In one example, D-glucose-6-phosphate was converted byglucose-6-phosphate-dehydrogenase to a oxidized byproduct with theconcomitant reduction of NADP⁺ to NADPH. Yamanaka et al., J. Am. Chem.Soc. 117:9095-96 (1995). The types of reaction centers which may beencapsulated in a sol-gel, as contemplated by the present invention,thus includes those compounds or materials that react with more than onereactant. For example, many enzymes contemplated by the presentinvention for encapsulation require coenzymes in addition to anysubstrate.

[0212] One important characteristic of any encapsulated reaction centeris the degree of loading of the matrix. For example, the degree ofloading may affect the reactivity of a reaction center in the sol-gelmatrix. It has been reported that the activity of trypsin appeared todecrease with increased loading levels. Levels of loading contemplatedby the present invention include 0.001, 0.01, 0.1, 1, 3, 5, 10, 15, 20weight percent reaction center(s) and/or any additives to the matrix.

[0213] In certain embodiments, the present invention contemplates usingas a reaction center enzymes or other biological materials that areisolated from, or otherwise substantially free of other cellularproteins. The term “substantially free of other cellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations thereaction center of interest having less than 20% (by dry weight)contaminating protein, and preferably having less than 5% contaminatingprotein. The term “purified” as used herein preferably means at least80% by dry weight, more preferably in the range of 95-99% by weight, andmost preferably at least 99.8% by weight, of biological macromoleculesof the same type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above. “Isolated” and“purified” do not encompass either natural materials in their nativestate or natural materials that have been separated into components(e.g., in an acrylamide gel) but not obtained either as pure (e.g.lacking contaminating proteins, or chromatography reagents such asdenaturing agents and polymers, e.g. acrylamide or agarose) substancesor solutions.

[0214] In certain embodiments, the present invention contemplates usingfor the reaction center human homologs of any of the enzymes or otherbiological materials described herein, as well as orthologs and paralogs(homologs) in other species. The term “ortholog” refers to proteinswhich are homologs via speciation, e.g., closely related and assumed tohave common descent based on structural and functional considerations.Orthologous proteins function as recognizably the same activity indifferent species. The term “paralog” refers to genes or proteins whichare homologs via gene duplication, e.g., duplicated variants of a genewithin a genome. See also Fritch Syst Zool 19:99-113 (1970).

[0215] In certain embodiments, the present invention contemplateshomologs of any naturally occurring enzymes. Further, the presentinvention contemplates modification of the structure of any enzyme toenhance therapeutic or prophylactic efficacy, or stability (e.g., exvivo shelf life). Such modified peptides may be produced, for instance,by amino acid substitution, deletion, or addition.

[0216] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are can be divided intofour families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. In similar fashion, theamino acid repertoire can be grouped as (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine,valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally be grouped separately as aliphatic-hydroxyl; (4)aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine,glutamine; and (6) sulfur -containing=cysteine and methionine. (see, forexample, Biochemistry (2nd ed., Stryer et al. eds. 1981). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog to any naturally occurring enzyme (e.g., functional in the sensethat the resulting polypeptide mimics the wild-type form) can be readilydetermined by assaying for activity. Polypeptides in which more than onereplacement has taken place can readily be tested in the same manner.

[0217] This invention further contemplates a method for generating setsof combinatorial mutants of any nucleic acid encoding for an enzyme usedas a reaction center, as well as truncation mutants, and is especiallyuseful for identifying potential variant sequences (e.g. homologs) thatare functional in modulating the enzymatic activity of interest. Thepurpose of screening such combinatorial libraries is, for example, toidentify novel polypeptides that may convert prodrugs to biologicallyactive agents. In certain embodiments, such novel polypeptides mayconvert prodrugs that naturally occurring enzymes do not, which maytherefore allow a particular prodrug to be used in the present inventionthat otherwise would not have been possible. In other embodiments, theprodrug of interest is converted only by an encapsulated, novelpolypeptide. As a result, no biologically active agent is produced invivo except by the encapsulated reaction center of the matrix. Such aspecificity difference may have value because prodrugs that becomecyotoxic agents may have reduced toxicity if they are stable in vivo.Site-directed mutagenesis has been used in ADEPT to prepare mutants ofcarboxypeptidase A, so that only the mutants and no naturally occurringenzyme converts selected prodrugs into corresponding cytotoxic agents.Smith et al. J. Biol. Chem. 272:15804-16 (1997). Even a single aminoacid change may be sufficient to affect the specificity. Thus,combinatorially-derived homologs can be generated to have an increasedpotency or different specificity relative to a naturally occurring formof an enzyme or other biological macromolecule.

[0218] In one aspect of this method, the amino acid sequences for apopulation of homologs for any enzyme, or other related proteins, arealigned, preferably to promote the highest homology possible. Such apopulation of variants can include, for example, homologs from one ormore species. Amino acids which appear at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences. In a preferred embodiment, the variegated library of variantsis generated by combinatorial mutagenesis at the nucleic acid level, andis encoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins containing the set of sequences therein.

[0219] There are many ways by which such libraries of potential homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential sequences. The synthesis of degenerate oligonucleotidesis well known in the art. See, for example, Narang Tetrahedron 39:3(1983); Itakura et al. Recombinant DNA, Proc 3rd Cleveland Sympos.Macromolecules 273-89 (ed. A G Walton, Amsterdam: Elsevier 1981);Itakura et al. Annu. Rev. Biochem. 53:323 (1984); Itakura et al. Science198:1056 (1984); Ike et al. Nucleic Acid Res. 11:477 (1983). Suchtechniques have been employed in the directed evolution of otherproteins. See, for example, Scott et al. Science 249:386-90 (1990);Roberts et al. PNAS 89:2429-33 (1992); Devlin et al. Science 249:404-06(1990); Cwirla et al. PNAS 87:6378-82 (1990); as well as U.S. Pat. Nos.5,223,409, 5,198,346, and 5,096,815.

[0220] Likewise, a library of coding sequence fragments can be providedfor an enzyme of interest as a reaction center in order to generate avariegated population of fragments for screening and subsequentselection of bioactive fragments. A variety of techniques are known inthe art for generating such libraries, including chemical synthesis. Inone embodiment, a library of coding sequence fragments can be generatedby (i) treating a double stranded PCR fragment of an coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule; (ii) denaturing the double stranded DNA; (iii) renaturingthe DNA to form double stranded DNA which can include sense/antisensepairs from different nicked products; (iv) removing single strandedportions from reformed duplexes by treatment with S1 nuclease; and (v)ligating the resulting fragment library into an expression vector. Bythis exemplary method, an expression library can be derived which codesfor N-terminal, C-terminal and internal fragments of various sizes.

[0221] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of homologs for any enzyme of interest as a reaction center.The most widely used techniques for screening large gene librariestypically comprises cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors, and expressing the combinatorial genes under conditions inwhich detection of a desired activity facilitates relatively easyisolation of the vector encoding the gene whose product was detected.

[0222] 5.4.1. Enzymes Used for ADEPT

[0223] Enzymes that have been used for ADEPT may generally be used asthe encapsulated reaction center in the present invention. Such enzymeswere originally chosen because they convert a prodrug into abiologically active agent, and they are thereby of use in the presentinvention as well. Some examples of such enzymes, and the conversionthat they catalyze, follow. Other examples may be found in U.S. Pat.Nos. 5,714,148; 5,660,829, 5,587,161; and 5,405,990. In the presentinvention, enzymes used in ADEPT need not necessarily be used with thesame prodrug as used in the ADEPT application.

[0224] In one aspect of the invention, a variety of peptidases, whichcleave amide bonds, may be used as the reaction center. Jungheim et al.,Chem. Rev. 94:1553-66 (1994). In one embodiment, carboxypeptidase G2 canbe used as the reaction center with prodrugs to cleave an amide bond.One biologically active agent is nitrogen mustard, which is analkylating agent. Carboxypeptidase G2 cleaves an amide bond of a prodrugto give the free nitrogen mustard and glutamic acid. Bagshawe, Br. J.Cancer 56:531 (1987); Bagshawe et al. Br. J. Cancer 58:700 (1988). Inanother embodiment, carboxypeptidase A from bovine pancrease can be usedas the reaction center with prodrugs to cleave an amide bond. In oneembodiment, the biologically active agent is methotrexate, which is usedfor cancer chemotherapy, and the prodrugs are α-peptides ofmethotrexate, e.g., Glu-α-L-Ala-methotrexate andL-Glu-L-Phe-methotrexate. Vitols et al., Pteridines 3:125 (1992);Kuefner et al., Biochem. 28:2288 (1989); Haenseler et al., Biochem.31:891 (1992).

[0225] In another embodiment, penicillin V amidase from Fusariumoxysporum can be used as the reaction center with prodrugs to deacylatean N-acyl amine. In several embodiments, the biologically active agentis doxorubicin or mephalan, which are anticancer agents, and theprodrugs are N-acyl derivatives of doxorubicin or mephalan. Kerr et al.Cancer Immun. Immunother. 31:202 (1990). In another embodiment,penicillin G amidase can be used as the reaction center with prodrugs tocleave phenylacetamides. In several embodiments, the biologically activeagent is doxorubicin or mephalan, and the prodrugs are phenylacetamidederivatives of doxorubicin or mephalan. Kerr et al. supra. In anotherembodiment, the biologically active agent is palytoxin, which is apotent cytotoxin, and the prodrug isN-[(4′-hydroxyphenyl)acetyl]palytoxin. Because palytoxin asserts itseffect extracellularly, it may be able to overcome the multidrugresistance phenotype.

[0226] In another embodiment, urokinase may be the reaction center.Puromycin and doxorubicin have been produced using this enzyme. WO91/09134. In another embodiment, a variety of β-lactamases, which cleavecertain amide bonds, may be used as the reaction center. In oneembodiment, a biologically active agent is produced by covalentlyattaching the agent to the C-3′ position of cephalosporin or aderivative of cephalosporin, whereupon hydrolization of the prodrug by aβ-lactamase, e.g., P99 enzyme derived from Enterobacter cloacae 265A orenzyme derived from B. cereus, produces the free agent. Biologicallyactive agents that have been covalently attached to cephalosporin or aderivative of cephalosporin in this manner include: methotrexate;5-fluorouracil, which is often used for the treatment of colon cancer;LY233425, a potent analogue of the anticancer agent vinblastine;desacetylvinblastine hydrazine, a potent vinca alkaloid; nitrogenmustard alkylating agents; thioguanine; doxorubicin; mitomycin C; andDACCP, a carboplatinum-based drug that is a potent antitumor agent.Jungheim et al. Chem. Rev. 94:1553-66 (1994); Meyer et al. Antibody,Immunoconjugates, Radiopharm. 3:66 (1990); Jungheim et al., Antibody,Immunoconjugates, Radiopharm. 4:228 (1991); Shepard et al., Biomed.Chem. Lett. 1:21 (1991); EP0382411A2; Alexander et al. Tetrahedron Lett.32:3269 (1991); EPo392745A2; Svensson et al. Bioconj. Chem. 3:176(1992); Vrudhula et al. Bioconj. Chem. 4:334 (1993); Hudyma et al.Biomed. Chem. Lett. 3:323 (1993); EP0484870A2; Junghein et al.Heterocycles 35:33 (1993); Hanessian et al. Can J. Chem. 71:896 (1993).

[0227] In another aspect of the invention, alkaline phosphotase can beused as the reaction center with prodrugs to remove phosphate fromorganic phosphates. Biologically active agents that are produced in thismanner include etoposide, mitomycin-derived agents, nitrogen mustardderived agents, and doxorubicin. Senter et al. Proc. Nat. Acad. Sci.U.S.A. 85:4842 (1988); Senter et al. Cancer Res. 49:5789 (1989); Senter,FASEB J. 4:188 (1990); Sahin et al. Cancer Res. 50:6944 (1990).

[0228] In another aspect of the invention, glycosidases, which cleave aglycosidic linkage may be used as the reaction center. In oneembodiment, β-glucuronidase is used as the reaction center to producebiologically active agents, including nitrogen mustard derived agents,daunomycin, adriamycin, epirubicin. Roffler et al. Biomed Pharmacol.42:2062 (1991); Wang et al. Cancer Res. 52:4484 (1992); Deonarain et al.Br. J. Cancer 70:786-94 (1994). In another embodiment, β-Glucosidaseconverts amygdalin into glucose, benzaldehyde, and hydrogen cyanide, atoxic species. In another embodiment, α-galactosidase is used as thereaction center to produce daunorubicin. Andrianomenjanahary et al.Biomed. Chem. Lett. 2:1093 (1992).

[0229] In another aspect of the invention, cytosine deaminase, whichconverts cytosine into uracil, may be used as the reaction center. Inone embodiment, cytosine deaminase isolated from Bakers' yeast is usedto produce the antitumor agent 5-fluroruracil from 5-fluorocytosine.Senter et al. Bioconj. Chem. 2:447 (1991). In another aspect of theinvention, nitroreductase, which requires the presence of a cofactorsuch as NADH, may be used as the reaction center. The enzyme has beenused to produce 5-aziridin-4-hydroxyamino-2-nitrobenzamide from5-aziridin-2,4-dinitrobenzamide. Knox et al. Cancer Metathesis Rev.12:195 (1993).

[0230] In another aspect of the invention, oxidases, which producereduced oxygen species, e.g., peroxide, superoxide, and hydroxylradicals, may be used as the reaction center. In one embodiment, glucoseoxidase and lactoperoxidase convert glucose and iodide into hydrogenperoxide and toxic iodine species. Ito et al. Bone Marrow Transplant.6:395-98 (1990); Stanislawski (1989). In another embodiment, xathineoxidase produces reduced oxygen species from either xanthine orhypoxanthine. Dinota et al. Bone Marrow Transplant. 6:31-36 (1990).

[0231] 5.4.2. Other Enzymes

[0232] In addition to the enzymes used for ADEPT, for which a prodrugmay have already been identified, other enzymes may be used as thereaction center. In using any enzyme in the present invention, it may benecessary to consider which compounds will be converted by the enzyme.Which enzyme is most suitable for encapsulation as the reaction centerdepends, in part, on the expected use of any matrix.

[0233] In deciding which enzyme may be appropriate for any application,the general classification of enzymes may be used in identifying andconsidering different types of reactions that a reaction center couldpossibly catalyze. These classes include: (i) oxidoreductases (acting onthe CH—OH group of donors; acting on the aldehyde or oxo group ofdonors; acting on the CH—CH group of donors; acting on the CH—NH(2)group of donors; acting on the CH—NH group of donors; acting on NADH orNADPH; acting on other nitrogenous compounds as donors; acting on asulfur group of donors; acting on a heme group of donors; acting ondiphenols and related substances as donors; acting on a peroxide asacceptor (peroxidases); acting on hydrogen as donor; acting on singledonors with incorporation of molecular oxygen; acting on paired donorswith incorporation of molecular oxygen; acting on superoxide radicals asacceptor; oxidizing metal ions; acting on —CH(2) groups; acting onreduced ferredoxin as donor; acting on reduced flavodoxin as donor;other oxidoreductases); (ii) transferases (transferring one-carbongroups; transferring aldehyde or ketone residues; acyltransferases;glycosyltransferases; transferring alkyl or aryl groups, other thanmethyl groups; transferring nitrogenous groups; transferringphosphorous-containing groups; transferring sulfur-containing groups;transferring selenium-containing groups); (iii) hydrolases (acting onester bonds; glycosidases; acting on ether bonds; acting on peptidebonds (peptide hydrolases); acting on carbon-nitrogen bonds, other thanpeptide bonds; acting on acid anhydrides; acting on carbon-carbon bonds;acting on halide bonds; acting on phosphorus-nitrogen bonds; acting onsulfur-nitrogen bonds; acting on carbon-phosphorus bonds; acting onsulfur-sulfur bonds); lyases (carbon-carbon lyases; carbon-oxygenlyases; carbon-nitrogen lyases; carbon-sulfur lyases; carbon-halidelyases; phosphorus-oxygen lyases; other lyases); (iv) isomerases(racemases and epimerases; cis-trans-isomerases; intramolecularoxidoreductases; intramolecular transferases (mutases); intramolecularlyases; other isomerases); (v) ligases (forming carbon-oxygen bonds;forming carbon-sulfur bonds; forming carbon-nitrogen bonds; formingcarbon-carbon bonds; forming phosphoric ester bonds).

[0234] Illustrative examples of enzymes that may serve as reactioncenters in the present invention include, without limitation: alcoholdehydrogenase (EC 1.1.1.1), homoserine dehydrogenase (EC 1.1.1.3),(R,R)-butanediol dehydrogenase (EC 1.1.1.4), glycerol dehydrogenase (EC1.1.1.6), glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8),D-xylulose reductase (EC 1.1.1.9), L-xylulose reductase (EC 1.1.1.10),L-iditol dehydrogenase (EC 1.1.1.14), mannitol-1-phosphate dehydrogenase(EC 1.1.1.17), myo-inositol 2-dehydrogenase (EC 1.1.1.18), aldehydereductase (EC 1.1.1.21), quinate dehydrogenase (EC 1.1.1.24), shikimatedehydrogenase (EC 1.1.1.25), glyoxylate reductase (EC 1.1.1.26),L-lactate dehydrogenase (EC 1.1.1.27), D-lactate dehydrogenase (EC1.1.1.28), glycerate dehydrogenase (EC 1.1.1.29), 3-hydroxybutyratedehydrogenase (EC 1.1.1.30), 3-hydroxyisobutyrate dehydrogenase (EC1.1.1.31), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), malatedehydrogenase (EC 1.1.1.37), malate dehydrogenase and aspartateaminotransferase (EC 1.1.1.37 & 2.6.1.1), malate dehydrogenlase andcitrate (si)-synthase (EC 1.1.1.37 and 4.1.3.7), malate dehydrogenase(decarboxylating) (EC 1.1.1.39), malate dehydrogenase(oxaloacetate-decarboxylating) (NADP+) (EC 1.1.1.40), isocitratedehydrogenase (NADP+) (EC 1.1.1.42), phosphogluconate dehydrogenase(decarboxylating) (EC 1.1.1.44), glucose dehydrogenase (EC 1.1.1.47),galactose dehydrogenase (EC 1.1.1.48), glucose-6-phosphate dehydrogenase(EC 1.1.1.49), glucose-6-phosphate dehydrogenase and6-phosphogluconolactonase (EC 1.1.1.49 & 3.1.1.31), 3-hydroxysteroiddehydrogenase (EC 1.1.1.50), 3(or 17)-hydroxysteroid dehydrogenase (EC1.1.1.51), lactaldehyde reductase (NADPH) (EC 1.1.1.55), ribitoldehydrogenase (EC 1.1.1.56), 3-hydroxypropionate dehydrogenase (EC1.1.1.59), 2-hydroxy-3-oxopropionate reductase (EC 1.1.1.60),4-hydroxybutyrate dehydrogenase (EC 1.1.1.61), estradiol17-dehydrogenase (EC 1.1.1.62), mannitol dehydrogenase (EC 1.1.1.67),gluconate 5-dehydrogenase (EC 1.1.1.69), glycerol dehydrogenase (NADP+)(EC 1.1.1.72), glyoxylate reductase (NADP+) (EC 1.1.1.79), aryl-alcoholdehydrogenase (EC 1.1.1.90), phosphoglycerate dehydrogenase (EC1.1.1.95), diiodophenylpyruvate reductase (EC 1.1.1.96),3-hydroxybenzyl-alcohol dehydrogenase (EC 1.1.1.97),3-oxoacyl-[acyl-carrier-protein] reductase (EC 1.1.1.100), carnitinedehydrogenase (EC 1.1.1.108), indolelactate dehydrogenase (EC1.1.1.110), glucose dehydrogenase (NADP+) (EC 1.1.1.119), fructose5-dehydrogenase (NADP+) (EC 1.1.1.124), 2-deoxy-D-gluconatedehydrogenase (EC 1.1.1.125), L-threonate dehydrogenase (EC 1.1.1.129),sorbitol-6-phosphate dehydrogenase (EC 1.1.1.140),15-hydroxyprostaglandin dehydrogenase (NAD+) (EC 1.1.1.141),21-hydroxysteroid dehydrogenase (NAD+) (EC 1.1.1.150), sepiapterinreductase (EC 1.1.1.153), coniferyl-alcohol dehydrogenase (EC1.1.1.194), (R)-2-hydroxyglutarate dehydrogenase (EC 1.1.1.a),sorbitol-6-phosphate dehydrogenase (NADP+) (EC 1.1.1.b), gluconate2-dehydrogenase (EC 1.1.99.3), lactate-malate transhydrogenase (EC1.1.99.7), glucoside 3-dehydrogenase (EC 1.1.99.13), formatedehydrogenase (EC 1.2.1.2), acetaldehyde dehydrogenase (acetylating) (EC1.2.1.10), aspartate-semialdehyde dehydrogenase (EC 1.2.1.11),glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12),glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (EC1.2.1.12 & 2.7.2.3), glyoxylate dehydrogenase (acylating) (EC 1.2.1.17),formate dehydrogenase (NADP+) (EC 1.2.1.43), succinate dehydrogenase (EC1.3.99.1), butyryl-CoA dehydrogenase (EC 1.3.99.2), dihydroorotatedehydrogenase (EC 1.3.99.11), alanine dehydrogenase (EC 1.4.1.1),glutamate dehydrogenase (EC 1.4.1.2), glutamate dehydrogenase (NAD(P)+)(EC 1.4.1.3), glutamate dehydrogenase (NADP+) (EC 1.4.1.4), leucinedehydrogenase (EC 1.4.1.9), glycine dehydrogenase (EC 1.4.1.10),L-erythro-3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11),2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12),pyrroline-2-carboxylate reductase (EC 1.5.1.1), pyrroline-5-carboxylatereductase (EC EC 1.5.1.2), dihydrofolate reductase (EC EC 1.5.1.3),methylenetetrahydrofolate dehydrogenase (NADP+) (EC 1.5.1.5), D-octopinedehydrogenase (EC 1.5.1.11), methylenetetrahydrofolate dehydrogenase(NAD+) (EC 1.5.1.15), alanopine dehydrogenase (EC 1.5.1.17),1-piperidine-2-carboxylate reductase (EC 1.5.1.21), NAD(P)+transhydrogenase (EC 1.6.1.1), glutathione reductase (NAD(P)H) (EC1.6.4.2), thioredoxin reductase (NADPH) (EC 1.6.4.5), NADH dehydrogenase(EC 1.6.99.3), 5,10-methylenetetrahydrofolate reductase (FADH2) (EC1.7.99.5), dihydrolipoamide dehydrogenase (EC 1.8.1.4),glutathione-CoA-glutathione transhydrogenase (EC 1.8.4.3), cytochrome-coxidase (EC EC 1.9.3.1), hydrogen dehydrogenase (EC 1.12.1.2),thetin-homocysteine S-methyltransferase (EC 2.1.1.3), homocysteineS-methyltransferase (EC 2.1.1.10), thymidylate synthase (EC 2.1.1.45),glycine hydroxymethyltransferase (EC 2.1.2.1), glycineformiminotransferase (EC 2.1.2.4), glutamate formiminotransferase (EC2.1.2.5), D-alanine 2-hydroxymethyltransferase (EC 2.1.2.7),aminomethyltransferase (EC 2.1.2.10), methylmalonyl-CoAcarboxyltransferase (EC 2.1.3.1), omithine carbamoyltransferase (EC2.1.3.3), oxamate carbamoyltransferase (EC 2.1.3.5), glycineamidinotransferase (EC 2.1.4.1), transketolase (EC 2.2.1.1),transaldolase (EC 2.2.1.2), imidazole N-acetyltransferase and phosphateacetyltransferase (EC 2.3.1.2 & 2.3.1.8), arylamine N-acetyltransferase(EC 2.3.1.5), choline O-acetyltransferase (EC 2.3.1.6), camitineO-acetyltransferase (EC 2.3.1.7), phosphate acetyltransferase (EC2.3.1.8), phosphate acetyltransferase and formate C-acetyltransferase(EC 2.3.1.8 & 2.3.1.54), phosphate acetyltransferase and acetate kinase(EC 2.3.1.8 & 2.7.2.1), acetyl-CoA C-acetyltransferase (EC 2.3.1.9),carnitine O-palmitoyltransferase (EC 2.3.1.21), glutarnateN-acetyltransferase (EC 2.3.1.35), [acyl-carrier-protein]S-acetyltransferase (EC 2.3.1.38), [acyl-carrier-protein]S-malonyltransferase (EC 2.3.1.39), formate C-acetyltransferase (EC2.3.1.54), sucrose phosphorylase (EC 2.4.1.7), maltose phosphorylase (EC2.4.1.8), levansucrase (EC 2.4.1.10), sucrose synthase (EC 2.4.1.13),sucrose-phosphate synthase (EC 2.4.1.14),,-trehalose-phosphate synthase(UDP-forming) (EC 2.4.1.15), cellobiose phosphorylase (EC 2.4.1.20),laminaribiose phosphorylase (EC 2.4.1.31),,-trehalose phosphorylase (EC2.4.1.64), galactinol-raffinose galactosyltransferase (EC 2.4.1.67),sinapate 1-glucosyltransferase (EC 2.4.1.120), purine-nucleosidephosphorylase (EC 2.4.2.1), purine-nucleoside phosphorylase andpyrimidine-nucleoside phosphorylase (EC 2.4.2.1 & 2.4.2.2),pyrimidine-nucleoside phosphorylase (EC 2.4.2.2), uridine phosphorylase(EC 2.4.2.3), nucleoside deoxyribosyltransferase (EC 2.4.2.6), adeninephosphoribosyltransferase (EC 2.4.2.7), hypoxanthinephosphoribosyltransferase (EC 2.4.2.8), orotatephosphoribosyltransferase (EC 2.4.2.10), guanosine phosphorylase (EC2.4.2.15), thiamine pyridinylase (EC 2.5.1.2), thiamin-phosphatepyrophosphorylase (EC 2.5.1.3), aspartate transaminase (EC 2.6.1.1),malate dehydrogenase and aspartate transaminase (EC 1.1.1.37 & 2.6.1.1),alanine transaminase (EC 2.6.1.2), histidinol-phosphate transaminase (EC2.6.1.9), omithine-oxo-acid transaminase (EC 2.6.1.13),glutamine-pyruvate aminotransaminase (EC 2.6.1.15),succinyldiaminopimelate transaminase (EC 2.6.1.17), -alanine-pyruvatetransaminase (EC 2.6.1.18), 4-aminobutyrate transaminase (EC 2.6.1.19),D-alanine transaminase (EC 2.6.1.21), pyridoxamine-pyruvate transaminase(EC 2.6.1.30), dTDP-4-amino-4,6-dideoxy-D-glucose transaminase (EC2.6.1.33), glycine-oxaloacetate transaminase (EC 2.6.1.35),2-aminoadipate transaminase (EC 2.6.1.39), serine-pyruvate transaminase(EC 2.6.1.51), phosphoserine transaminase (EC 2.6.1.52), hexokinase (EC2.7.1.1), galactokinase (EC 2.7.1.6), 6-phosphofructokinase (EC2.7.1.11), NAD+ kinase (EC 2.7.1.23), dephospho-CoA kinase (EC2.7.1.24), glycerol kinase (EC 2.7.1.30), protein kinase (EC 2.7.1.37),pyruvate kinase (EC 2.7.1.40), 1-phosphatidylinositol kinase (EC2.7.1.67), pyrophosphate-serine phosphotransferase (EC 2.7.1.80),pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90),acetate kinase (EC 2.7.2.1), carbamate kinase (EC 2.7.2.2),phosphoglycerate kinase (EC 2.7.2.3), glyceraldehyde-3-phosphatedehydrogenase and phosphoglycerate kinase (EC 1.2.1.12 & 2.7.2.3),aspartate kinase (EC 2.7.2.4), guanidinoacetate kinase (EC 2.7.3.1),creatine kinase (EC 2.7.3.2), creatine kinase and myosin ATPase (EC2.7.3.2 & 3.6.1.32), arginine kinase (EC 2.7.3.3), taurocyamine kinase(EC 2.7.3.4), lombricine kinase (EC 2.7.3.5), phosphomevalonate kinase(EC 2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase(EC 2.7.4.4), nucleoside-diphosphate kinase (EC 2.7.4.6), guanylatekinase (EC 2.7.4.8), nucleoside-triphosphate-adenylate kinase (EC2.7.4.10), (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13), cytidylatekinase (EC 2.7.4.14), ribose-phosphate pyrophosphokinase (EC 2.7.6.1),nicotinamide-nucleotide adenylyltransferase (EC 2.7.7.1), sulfateadenylyltransferase (EC 2.7.7.4), sulfate adenylyltransferase andinorganic pyrophosphatase (EC 2.7.7.4 & 3.6.1.1), DNA-directed DNApolymerase (EC 2.7.7.7), UTP-glucose-1-phosphate uridylyltransferase (EC2.7.7.9), UDPglucose-hexose-1-phosphate unidylyltransferase (EC2.7.7.12), UDPglucose-hexose 1-phosphate uridylyltransferase andUDPglucose (EC 2.7.7.12 & 5.1.3.2), mannose-1-phosphateguanylyltransferase (EC 2.7.7.13), ethanolamine-phosphatecytidylyltransferase (EC 2.7.7.14), choline-phosphatecytidylyltransferase (EC 2.7.7.15), UDP-N-acetylglucosaminepyrophosphorylase (EC 2.7.7.23), glucose-1-phosphatethymidylyltransferase (EC 2.7.7.24), glucose-1-phosphateadenylyltransferase (EC 2.7.7.27), glucose-1-phosphatecytidylyltransferase (EC 2.7.7.33), glucose-1-phosphateguanylyltransferase (EC 2.7.7.34), [glutamate-ammonia-ligase]adenylyltransferase (EC 2.7.7.42), glucuronate-1-phosphateuridylyltransferase (EC 2.7.7.44), pyruvate, orthophosphate dikinase (EC2.7.9.1), aryl sulfotransferase (EC 2.8.2.1), 3-oxoacid CoA-transferase(EC 2.8.3.5), acetate CoA-transferase (EC 2.8.3.8), triacylglycerollipase (EC 3.1.1.3), acetylcholinesterase (EC 3.1.1.7),retinyl-palmitate esterase (EC 3.1.1.21), glucose-6-phosphatedehydrogenase and 6-phosphogluconolactonase (EC 1.1.1.49 & 3.1.1.31),alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2),phosphoserine phosphatase (EC 3.1.3.3), 5′-nucleosidase (EC 3.1.3.5),fructose-biphosphatase (EC 3.1.3.11), phosphodiesterase I (EC 3.1.4.1),3′,5′-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17),phosphohydrolase (unclassified) (EC 3.1.4.a), ribonuclease T2 (EC3.1.27.1), pancreatic ribonuclease (EC 3.1.27.5), ribonuclease(unclassified) (EC 3.1.27.a), cyclomaltodextrin glucanotransferase and-amylase (EC 2.4.1.19 & 3.2.1.1), -amylase (EC 3.2.1.2), glucan1,4--glucosidase (EC 3.2.1.3), oligo-1,6-glucosidase (EC 3.2.1.10),-glucosidase (EC 3.2.1.20), -glucosidase (EC 3.2.1.21), -galactosidase(EC 3.2.1.23), -mannosidase (EC 3.2.1.24), -fructofuranosidase (EC3.2.1.26), -dextrin endo-1,6-glucosidase (EC 3.2.1.41), AMP nucleosidase(EC 3.2.2.4), NAD+ nucleosidase (EC 3.2.2.5), NAD(P)+ nucleosidase (EC3.2.2.6), adenosine nucleosidase (EC 3.2.2.7), adenosylhomocysteinase(EC 3.3.1.1), leucyl aminopeptidase (EC 3.4.11.1), dipeptidyl-peptidaseI (EC 3.4.14.1), carboxypeptidase A (EC 3.4.17.1), gly-Xcarboxypeptidase (EC 3.4.17.4), -glu-X carboxypeptidase (EC 3.4.19.9),chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), papain (EC 3.4.22.2),pepsin A (EC 3.4.23.1), chymosin (EC 3.4.23.4), thermolysin (EC3.4.24.27), asparaginase (EC 3.5.1.1), glutaminase (EC 3.5.1.2), urease(EC 3.5.1.5), penicillin amidase (EC 3.5.1.11), aminoacylase (EC3.5.1.14), pantothenase (EC 3.5.1.22), N-methyl-2-oxoglutaramatehydrolase (EC 3.5.1.36), dihydroorotase (EC 3.5.2.3),carboxymethylhydantoinase (EC 3.5.2.4), -lactamase (EC 3.5.2.6),arginase (EC 3.5.3.1), allantoicase (EC 3.5.3.4), arginine deiminase (EC3.5.3.6), adenosine deaminase (EC 3.5.4.4), cytidine deaminase (EC3.5.4.5), AMP deaminase (EC 3.5.4.6), methenyltetrahydrofolatecyclohydrolase (EC 3.5.4.9), inorganic pyrophosphatase (EC 3.6.1.1),sulfate adenylyltransferase and inorganic pyrophosphatase (EC 2.7.7.4 &3.6.1.1), trimetaphosphatase (EC 3.6.1.2), nucleotide pyrophosphatase(EC 3.6.1.9), myosin ATPase (EC 3.6.1.32), creatine kinase and myosinATPase (EC 2.7.3.2 & 3.6.1.32), Ca2+-transporting ATPase (EC 3.6.1.38),chymotrypsin (EC 3.4.21.1), thermolysin (EC 3.4.24.4),phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32),phosphoenolpyruvate carboxykinase (diphosphate) (EC 4.1.1.38),ribulose-biphosphate carboxylase (EC 4.1.1.39), ketotetraose-phosphatealdolase (EC 4.1.2.2), deoxyribose-phosphate aldolase (EC 4.1.2.4),fructose-biphosphate aldolase (EC 4.1.2.13), fructose-biphosphatealdolase and triose-phosphate isomerase (EC 4.1.2.13 & 5.3.1.1),2-dehydro-3-deoxyphosphogluconate aldolase (EC 4.1.2.14),L-fuculose-phosphate aldolase (EC 4.1.2.17),2-dehydro-3-deoxy-L-pentonate aldolase (EC 4.1.2.18),rhamnulose-1-phosphate aldolase (EC 4.1.2.19),2-dehydro-3-deoxy-6-phosphogalactonate aldolase (EC 4.1.2.21),D-arabino-3-hexulose phosphate formaldehyde lyase (EC 4.1.2.a),isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2),N-acetylneuraminate lyase (EC 4.1.3.3), citrate (pro-3S)-lyase (EC4.1.3.6), citrate (si)-synthase (EC 4.1.3.7), citrate (si)-synthase andmalate dehydrogenase (EC 4.1.3.7 & 1.1.1.37), ATP citrate(pro-3S)-lyase(EC 4.1.3.8), 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16),citramalate lyase (EC 4.1.3.22), malyl-CoA lyase (EC 4.1.3.24),2,3-dimethylmalate lyase (EC 4.1.3.32), tryptophanase (EC 4.1.99.1),fumarate hydratase (EC 4.2.1.2), aconitate hydratase (EC 4.2.1.3),3-dehydroquinate dehydratase (EC 4.2.1.10), phosphopyruvate hydratase(EC 4.2.1.11), enoyl-CoA hydratase (EC 4.2.1.17), tryptophan synthase(EC 4.2.1.20), maleate hydratase (EC 4.2.1.31), (S)-2-methylmalatedehydratase (EC 4.2.1.34), (R)-2-methylmalate dehydratase (EC 4.2.1.35),D-glutamate cyclase (EC 4.2.1.48), urocanate hydratase (EC 4.2.1.49),crotonoyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58),dimethylmaleate hydratase (EC 4.2.1.85), 3-hydroxybutyryl-CoA dehyratase(EC 4.2.1.a), aspartate ammonia-lyase (EC 4.3.1.1), methylaspartateammonia-lyase (EC 4.3.1.2), histidine ammonia-lyase (EC 4.3.1.3),phenylalanine ammonia-lyase (EC 4.3.1.5), -alanyl-CoA ammonia lyase (EC4.3.1.6), arginosuccinate lyase (EC 4.3.2.1), adenylosuccinate lyase (EC4.3.2.2), ureidoglycolate lyase (EC 4.3.2.3), lactoylglutathione lyase(EC 4.4.1.5), adenylate cyclase (EC 4.6.1.1), alanine racemase (EC5.1.1.1), glutamate racemase (EC 5.1.1.3), lysine racemase (EC 5.1.1.5),diaminopimelate epimerase (EC 5.1.1.7), 4-hydroxyproline epimerase (EC5.1.1.8), amino-acid racemase (EC 5.1.1.10), ribulose-phosphate3-epimerase (EC 5.1.3.1), UDPglucose 4-epimerase (EC 5.1.3.2),UDPglucose 4-epimerase and UDPglucose-hexose 1-phosphate (EC 5.1.3.2 &2.7.7.12), L-ribulose-phosphate 4-epimerase (EC 5.1.3.4), UDParabinose4-epimerase (EC 5.1.3.5), UDPglucuronate 4-epimerase (EC 5.1.3.6),N-acylglucosamine 2-epimerase (EC 5.1.3.8),N-acylglucosamine-6-phosphate 2-epimerase (EC 5.1.3.9), CDPabequoseepimerase (EC 5.1.3.10), glucose-6-phosphate 1-epimerase (EC 5.1.3.15),GDP-D-mannose 3,5-epimerase (EC 5.1.3.18), methylmalonyl-CoA epimerase(EC 5.1.99.1), retinal isomerase (EC 5.2.1.3), linoleate isomerase (EC5.2.1.5), triose-phosphate isomerase (EC 5.3.1.1), triose-phosphateisomerase and fructose-bisphosphate aldolase (EC 5.3.1.1 & 4.1.2.13),erythrose isomerase (EC 5.3.1.2), arabinose isomerase (EC 5.3.1.3),L-arabinose isomerase (EC 5.3.1.4), xylose isomerase (EC 5.3.1.5),ribose-5-phosphate isomerase (EC 5.3.1.6), mannose isomerase (EC5.3.1.7), mannose-6-phosphate isomerase (EC 5.3.1.8),glucose-6-phosphate isomerase (EC 5.3.1.9), glucosamine-6-phosphateisomerase (EC 5.3.1.10), glucuronate isomerase (EC 5.3.1.12),arabinose-5-phosphate isomerase (EC 5.3.1.13), L-rhamnose isomerase (EC5.3.1.14), D-lyxose ketol-isomerase (EC 5.3.1.15), ribose isomerase (EC5.3.1.20), L-mannose ketol-isomerase (EC 5.3.1.a),phospho-3-hexuloisomerase (EC 5.3.1.b), phenylpyruvate tautomerase (EC5.3.2.1), oxaloacetate tautomerase (EC 5.3.2.2), isopentenyl-diphosphate-isomerase (EC 5.3.3.2), methylitaconate -isomerase (EC 5.3.3.6),phosphoglycerate mutase (EC 5.4.2.1), phosphoglucomutase (EC 5.4.2.2),phosphoacetylglucosamine mutase (EC 5.4.2.3), -phosphoglucomutase (EC5.4.2.6), phosphopentomutase (EC 5.4.2.7), phosphomannomutase (EC5.4.2.8), lysine 2,3-aminomutase (EC 5.4.3.2), D-omithine4,5-aminomutase (EC 5.4.3.5), methylaspartate mutase (EC 5.4.99.1),methylmalonyl-CoA mutase (EC 5.4.99.2), 2-methyleneglutarate mutase (EC5.4.99.4), muconate cycloisomerase (EC 5.5.1.1), tetrahydroxypteridinecycloisomerase (EC 5.5.1.3), chalcone isomerase (EC 5.5.1.6),valine-tRNA ligase (EC 6.1.1.9), acetate-CoA ligase (EC 6.2.1.1),butyrate CoA ligase (EC 6.2.1.2), succinate-CoA ligase (GDP-forming) (EC6.2.1.4), succinate-CoA ligase (ADP forming) (EC 6.2.1.5),glutamate-ammonia ligase (EC 6.3.1.2), formate-tetrahydrofolate ligase(EC 6.3.4.3), adenylosuccinate synthase (EC 6.3.4.4), arginosuccinatesynthase (EC 6.3.4.5), pyruvate carboxylase (EC 6.4.1.1), propanoyl-CoAcarboxylase (EC 6.4.1.3), hemoglobin, tyrosine hydroxylase, prohormoneconvertase, bcl-2, dopa decarboxylase, and dopamine beta-hydroxylase.

[0235] 5.4.3. Assay of Encapsulated Reaction Centers

[0236] Any number of methods are available to determine whether areaction center retains its ability to convert prodrug to biologicallyactive agent upon encapsulation. Those of skill in the art will be ableto modify, if necessary, any standard procedures developed for assayingthe reaction center in free solution for assaying the encapsulatedreaction center. For example, if the matrix is transparent, as is truefor silica-based sol-gel matrices, then standard visible and UV-Vistechniques for solid materials may be employed. Yamanka et al. J.Sol-Gel sci. & tech. 7:117-21 (1996). If the reaction center is redoxactive center, e.g., a transition metal, then other spectroscopies, suchas EPR, may be employed. Lin et al. J. Sol-Gel Sci. & Tech. 7:19-26(1996). As discussed below for the prodrugs, assaying for reactioncenter activity often involves measuring the reactants and products, andsolution techniques may be applicable. Alternatively, coenzymes,cofactors, or other reactants involved in any reaction center may bemonitored as an assay for activity of any encapsulated reaction center.

[0237] From such assays, it should be possible to determine the reactionkinetics for the encapsulated reaction center. In general, for enzymes,the reaction kinetics may correspond to the Michaelis-Menten treatment.Zubay et al. Biochemistry 137-141 (1983). An apparent Michaelis constant(Km) may be determined for the encapsulated reaction center. Dosoretz etal., J. Sol-Gel Sci. & Tech. 7:7-11 (1996). The Km for thenonencapsulated reaction center and the Km of the encapsulated reactioncenter may be compared. In certain embodiments, the ratio of Km(nonencapsulated) to Km (encapsulated) may be greater than one. Dosoretzet al., supra. In other embodiments, the ratio may be less than one.Venton et al. Biochim Biophys Acta 1250:117-25 (1995). The presentinvention contemplates ratios of 100, 10, 5, 1, 0.5, 0.1, 0.005, 0.01,and 0.001. Of course, for determining any of these ratios, theconditions of the reaction should be kept as similar as possible.

[0238] The encapsulation of reaction centers allows for the design ofnovel assays for reaction center activity. For example, a secondreaction center may be encapsulated so as to help assay the activity ofa first encapsulated reaction center. Yamanka et al. reportencapsulating both oxalate oxidase and peroxidase. The peroxidaseconverts two dye precursors into a detectable dye using hydrogenperoxide, which is formed by oxalate oxidase from oxalate, water, anddioxygen. Yamanka et al. J. Sol-Gel Sci. & Tech. 7:117-21 (1996). Hence,the peroxidase in this sol-gel matrix assists in assaying the reactionkinetics of the oxalate oxidase. Yamanka et al. report that this enzymesystem is useful as a diagnostic for the decreased secretion of oxalatein cases of hyperglycinemia, hypoclycinuria, and hyperoxaluria. Ngo etal. Anal. Biochem. 105:389 (1980).

[0239] 5.5. Prodrugs

[0240] 5.5.1. Prodrugs Contemplated by the Invention

[0241] A variety of materials or compounds may be employed as prodrugsin the present invention. A number of such prodrugs have been discussedelsewhere, including when considering possible reaction centers and usesof the present invention. Any compound that is biologically active maybe used in the present invention as a prodrug, as long as a suitableprodrug may be prepared that may be converted by a reaction center intoa biologically active compound. The matrices of the present inventionmay be administered by-way of oral ingestion or implantation. Ifimplantation is desired, they can be implanted subcutaneously,constitute a part of a prosthesis, or be inserted in a cavity of thehuman body. Subcutaneous implantation using a syringe consists ofinjecting the amtrices directly into subcutaneous tissue. Thus, thematrices of the present invention can be suspended in a physiologicalbuffer and introduced via a syringe to the desired site. In certaincases, a biologically active agent may itself be used as a prodrug inthe present invention if a reaction center modulates its biologicalactivity upon reaction.

[0242] A number of considerations may be weighed by those of skill inthe art in determining which prodrug is appropriate for any use of thepresent invention. For example, it may be necessary to match a prodrugwith a reaction center that has a high activity for conversion of theprodrug. Alternatively, in choosing a prodrug, it may be important toconsider where in a subject the resulting matrix may be administered,e.g., the use of the prodrug L-dopa to produce the biologically activeagent dopamine in the striatum. Another possible consideration may bethe physical dimension of any prodrug, for to operate as a prodrug, itmay need to diffuse into the matrix for conversion by the reactioncenter to a biologically active agent. (Alternatively, the reactioncenter may be located on the surface of the matrix, whereupon nodiffusion is necessary for conversion of the prodrug.) However, evenantibodies have been observed to diffuse into matrices of the presentinvention, so any prodrug of at least that dimension may be used in thepresent invention. As discussed in preparing the matrices of the presentinvention, it may be necessary to ensure that the physical size of thereaction center is greater than that of its counterpart prodrug so as toprevent leaching. This criteria need not always apply, however, becausefor example, the reaction center may be covalently attached to thematrix, which may prevent any substantial leaching, or alternatively,any leaching that may occur may be acceptable for any use that thematrix is put.

[0243] Possible biologically active agents, which may be used asprodrugs in the present invention after appropriate modification,include without limitation, medicaments; vitamins; mineral supplements;substances used for the treatment, prevention, diagnosis, cure ormitigation of disease or illness; or substances which affect thestructure or function of the body.

[0244] Specific types of biologically active agents include, withoutlimitation: anti-angiogenesis factors, antiinfectives such asantibiotics and antiviral agents; analgesics and analgesic combinations;anorexics; antihelmintics; antiarthritics; antiasthmatic agents;anticonvulsants; antidepressants; antidiuretic agents; antidiarrheals;antihistamines; antiinflammatory agents; antimigraine preparations;antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics;antipsychotics; antipyretics, antispasmodics; anticholinergics;sympathomimetics; xanthine derivatives; cardiovascular preparationsincluding calcium channel blockers and beta-blockers such as pindololand antiarrhythmics; antihypertensives; catecholamines; diuretics;vasodilators including general coronary, peripheral and cerebral;central nervous system stimulants; cough and cold preparations,including decongestants; growth factors, hormones such as estradiol andother steroids, including corticosteroids; hypnotics;immunosuppressives; muscle relaxants; parasympatholytics;psychostimulants; sedatives; and tranquilizers; and naturally derived orgenetically engineered proteins, polysaccharides, glycoproteins,lipoproteins, interferons, cytokines, chemotherapeutic agents and otheranti-neoplastics, antibiotics, anti-virals, anti-fungals,anti-inflammatories, anticoagulants, lymphokines, or antigenicmaterials.

[0245] To illustrate further, other types of biologically active agentsthat may be used as prodrugs upon appropriate modification if necessary,including peptide, proteins or other biopolymers, e.g., interferons,interleukins, tumor necrosis factor, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), glialcell line-derived neurotrophic factor (GDNF), cholinergicdifferentiation factor/Leukemia inhibitory factor (CDF/LIF), epidermalgrowth factor (EGF), insulin-like growth factor (IGF), basic fibroblastgrowth factor (bFGF), platelet-derived growth factor (PDGF),erythropoietin, growth hormone, Substance-P, neurotensin, insulin,erythropoietin, albumin, transferrin, and other protein biologicalresponse modifiers.

[0246] Other examples of biologically active agents that may be used asprodrugs in accordance with the present invention either directly orafter appropriate modification include acebutolol, acetaminophen,acetohydoxamic acid, acetophenazine, acyclovir, adrenocorticoids,allopurinol, alprazolam, aluminum hydroxide, amantadine, ambenonium,amiloride, aminobenzoate potassium, amobarbital, amoxicillin,amphetamine, ampicillin, androgens, anesthetics, anticoagulants,anticonvulsants-dione type, antithyroid medicine, appetite suppressants,aspirin, atenolol, atropine, azatadine, bacampicillin, baclofen,beclomethasone, belladonna, bendroflumethiazide, benzoyl peroxide,benzthiazide, benztropine, betamethasone, betha nechol, biperiden,bisacodyl, bromocriptine, bromodiphenhydramine, brompheniramine,buclizine, bumetanide, busulfan, butabarbital, butaperazine, caffeine,calcium carbonate, captopril, carbamazepine, carbenicillin, carbidopa &levodopa, carbinoxamine inhibitors, carbonic anhydsase, carisoprodol,carphenazine, cascara, cefaclor, cefadroxil, cephalexin, cephradine,chlophedianol, chloral hydrate, chlorambucil, chloramphenicol,chlordiazepoxide, chloroquine, chlorothiazide, chlorotrianisene,chlorpheniramine, 6X chlorpromazine, chlorpropamide, chlorprothixene,chlorthalidone, chlorzoxazone, cholestyramine, cimetidine, cinoxacin,clemastine, clidinium, clindamycin, clofibrate, clomiphere, clonidine,clorazepate, cloxacillin, colochicine, coloestipol, conjugated estrogen,contraceptives, cortisone, cromolyn, cyclacillin, cyclandelate,cyclizine, cyclobenzaprine, cyclophosphamide, cyclothiazide, cycrimine,cyproheptadine, danazol, danthron, dantrolene, dapsone,dextroamphetaminte, dexamethasone, dexchlorpheniramine,dextromethorphan, diazepan, dicloxacillin, dicyclomine,diethylstilbestrol, diflunisal, digitalis, diltiazen, dimenhydrinate,dimethindene, diphenhydramine, diphenidol, diphenoxylate & atrophive,diphenylopyraline, dipyradamole, disopyramide, disulfiram, divalporex,docusate calcium, docusate potassium, docusate sodium, doxyloamine,dronabinol ephedrine, epinephrine, ergoloidmesylates, ergonovine,ergotamine, erythromycins, esterified estrogens, estradiol, estrogen,estrone, estropipute, etharynic acid, ethchlorvynol, ethinyl estradiol,ethopropazine, ethosaximide, ethotoin, fenoprofen, ferrous fumarate,ferrous gluconate, ferrous sulfate, flavoxate, flecainide, fluphenazine,fluprednisolone, flurazepam, folic acid, furosemide, gemfibrozil,glipizide, glyburide, glycopyrrolate, gold compounds, griseofuwin,guaifenesin, guanabenz, guanadrel, guanethidine, halazepam, haloperidol,hetacillin, hexobarbital, hydralazine, hydrochlorothiazide,hydrocortisone (cortisol), hydroflunethiazide, hydroxychloroquine,hydroxyzine, hyoscyatmine, ibuprofen, indapamide, indomethacin, insulin,iofoquinol, iron-polysaccharide, isoetharine, isoniazid, isopropamideisoproterenol, isotretinoin, isoxsuprine, kaolin & pectin, ketoconazole,lactulose, levodopa, lincomycin liothyronine, liotrix, lithium,loperamide, lorazepam, magnesium hydroxide, magnesium sulfate, magnesiumtrisilicate, maprotiline, meclizine, meclofenamate, medroxyproyesterone,melenamic acid, melphalan, mephenytoin, mephobarbital, meprobamate,mercaptopurine, mesoridazine, metaproterenol, metaxalone,methamphetamine, methaqualone, metharbital, methenamine, methicillin,methocarbamol, methotrexate, methsuximide, methyclothinzide,methylcellulos, methyldopa, methylergonovine, methylphenidate,methylprednisolone, methysergide, metoclopramide, metolazone,metoprolol, metronidazole, minoxidil, mitotane, monamine oxidaseinhibitors, nadolol, nafcillin, nalidixic acid, naproxen, narcoticanalgesics, neomycin, neostigmine, niacin, nicotine, nifedipine,nitrates, nitrofurantoin, nomifensine, norethindrone, norethindroneacetate, norgestrel, nylidrin, nystatin, orphenadrine, oxacillin,oxazepam, oxprenolol, oxymetazoline, oxyphenbutazone, pancrelipase,pantothenic acid, papaverine, para-aminosalicylic acid, paramethasone,paregoric, pemoline, penicillamine, penicillin, penicillin -v,pentobarbital, perphenazine, phenacetin, phenazopyridine, pheniramine,phenobarbital, phenolphthalein, phenprocoumon, phensuximide,phenylbutazone, phenylephrine, phenylpropanolamine, phenyl toloxamine,phenytoin, pilocarpine, pindolol, piper acetazine, piroxicam, poloxamer,polycarbophil calcium, polythiazide, potassium supplements, pruzepam,prazosin, prednisolone, prednisone, primidone, probenecid, probucol,procainamide, procarbazine, prochlorperazine, procyclidine, promazine,promethazine, propantheline, propranolol, pseudoephedrine, psoralens,psyllium, pyridostigmine, pyrodoxine, pyrilamine, pyrvinium, quinestrol,quinethazone, quinidine, quinine, ranitidine, rauwolfia alkaloids,riboflavin, rifampin, ritodrine, salicylates, scopolamine, secobarbital,senna, sannosides a & b, simethicone, sodium bicarbonate, sodiumphosphate, sodium fluoride, spironolactone, sucrulfate, sulfacytine,sulfamethoxazole, sulfasalazine, sulfinpyrazone, sulfisoxazole,sulindac, talbutal, tamazepam, terbutaline, terfenadine, terphinhydrate,teracyclines, thiabendazole, thiamine, thioridazine, thiothixene,thyroblobulin, thyroid, thyroxine, ticarcillin, timolol, tocainide,tolazamide, tolbutamide, tolmetin trozodone, tretinoin, triamcinolone,trianterene, triazolam, trichlormethiazide, tricyclic antidepressants,tridhexethyl, trifluoperazine, triflupromazine, trihexyphenidyl,trimeprazine, trimethobenzamine, trimethoprim, tripclennamine,triprolidine, valproic acid, verapamil, vitamin A, vitamin B-12, vitaminC, vitamin D, vitamin E, vitamin K, xanthine, parathyroid hormone,enkephalins, and endorphins.

[0247] To illustrate further, antimetabolites may be used as prodrugsupon appropriate modification if necessary, including without limitationmethotrexate, 5-fluorouracil, cytosine arabinoside (ara-C),5-azacytidine, 6-mercaptopurine, 6-thioguanine, and fludarabinephosphate. Antitumor antibiotics may include but are not limited todoxorubicin, daunorubicin, dactinomycin, bleomycin, mitomycin C,plicamycin, idarubicin, and mitoxantrone. Vinca alkaloids andepipodophyllotoxins may include, but are not limited to vincristine,vinblastine, vindesine, etoposide, and teniposide. Nitrosoureas,including carmustine, lomustine, semustine and streptozocin, may also beprodrugs, upon appropriate modification if necessary. Hormonaltherapeutics may also be prodrugs, upon appropriate modification ifnecessary, such as corticosteriods (cortisone acetate, hydrocortisone,prednisone, prednisolone, methyl prednisolone and dexamethasone),estrogens, (diethylstibesterol, estradiol, esterified estrogens,conjugated estrogen, chlorotiasnene), progestins (medroxyprogesteroneacetate, hydroxy progesterone caproate, megestrol acetate),antiestrogens (tamoxifen), aromastase inhibitors (aminoglutethimide),androgens (testosterone propionate, methyltestosterone, fluoxymesterone,testolactone), antiandrogens (flutamide), LHRH analogues (leuprolideacetate), and endocrines for prostate cancer (ketoconazole). Antitumordrugs that are radiation enhancers may also be used as prodrugs, uponappropriate modification if necessary. Examples of such biologicallyactive agents include, for example, the chemotherapeutic agents5′-fluorouracil, mitomycin, cisplatin and its derivatives, taxol,bleomycins, daunomycins, and methamycins. Antibiotics may be used asprodrugs as well, upon appropriate modification if necessary, and theyare well known to those of skill in the art, and include, for example,penicillins, cephalosporins, tetracyclines, ampicillin, aureothicin,bacitracin, chloramphenicol, cycloserine, erythromycin, gentamicin,gramacidins, kanamycins, neomycins, streptomycins, tobramycin, andvancomycin.

[0248] Other prodrugs, upon appropriate modification if necessary, whichmay be used in the present invention include those presently classifiedas investigational drugs, and can include, but are not limited toalkylating agents such as Nimustine AZQ, BZQ, cyclodisone, DADAG,CB10-227, CY233, DABIS maleate, EDMN, Fotemustine, Hepsulfam,Hexamethylmelamine, Mafosamide, MDMS, PCNU, Spiromustine, TA-077, TCNUand Temozolomide; antimetabolites, such as acivicin, Azacytidine,5-aza-deoxycytidine, A-TDA, Benzylidene glucose, Carbetimer, CB3717,Deazaguanine mesylate, DODOX, Doxifluridine, DUP-785, 10-EDAM,Fazarabine, Fludarabine, MZPES, MMPR, PALA, PLAC, TCAR, TMQ, TNC-P andPiritrexim; antitumor antibodies, such as AMPAS, BWA770U, BWA773U,BWA502U, Amonafide, m-AMSA, CI-921, Datelliptium, Mitonafide,Piroxantrone, Aclarubicin, Cytorhodin, Epirubicin, esorubicin,Idarubicin, Iodo-doxorubicin, Marcellomycin, Menaril, Morpholinoanthracyclines, Pirarubicin, and SM-5887; microtubule spindleinhibitors, such as Amphethinile, Navelbine, and Taxol; thealkyl-lysophospholipids, such as BM41-440, ET-18-OCH3, andHexacyclophosphocholine; metallic compounds, such as Gallium Nitrate,CL286558, CL287110, Cycloplatam, DWA2114R, NK121, Iproplatin,Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium compounds; andnovel compounds such as, for example, Aphidoicolin glycinate, Ambazone,BSO, Caracemide, DSG, Didemnin, B, DMFO, Elsamicin, Espertatrucin,Flavone acetic acid, HMBA, HHT, ICRF-187, Iododeoxyuridine, Ipomeanol,Liblomycin, Lonidamine, LY186641, MAP, MTQ, Merabarone SK&F104864,Suramin, Tallysomycin, Teniposide, THU and WR2721; and Toremifene,Trilosane, and zindoxifene.

[0249] 5.5.2. Assay's and Identification of Prodrugs

[0250] As a general matter, it will be clear to one of skill in the artwhich prodrugs may be used with which reaction centers so as to effectthe any of the uses of the subject invention, e.g., producing abiologically active agent. Prodrugs that display desiredcharacteristics, e.g., certain kinetic profiles of conversion of aprodrug by a reaction center to the corresponding biologically activeagent, may serve as lead compounds for the discovery of more desirableprodrugs.

[0251] In general, there are a number of methods by which usefulprodrugs of any reaction center encapsulated in a sol gel may bedetermined. For example, prodrugs may be individually prepared andtested for production of the corresponding biologically active agentupon interaction with the reaction center, whether encapsulated or not.

[0252] In another embodiment of the present invention, the use ofprodrugs in this invention readily lends itself to the creation ofcombinatorial libraries of compounds for screening prospective prodrugswith any particular reaction center or group of reaction centers toidentify prodrugs of such reaction centers. For the purposes of thepresent invention, the application of combinatorial chemistry may beespecially valuable because it may render identification of a suitableprodrug of a biologically active agent for use with a particularreaction center more facile. A combinatorial library for the purposes ofthe present invention is a mixture of chemically related compounds whichmay be screened together for a desired property, e.g., conversion by aparticular reaction center or reaction centers to produce a biologicallyactive agent. Such libraries may be in solution or covalently linked toa solid support. The preparation of many related compounds asprospective prodrugs in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate reactivity of any prospective prodrug maybe done by conventional methods.

[0253] For purposes of this invention, diversity in a library may becreated at a variety of different levels. In general, for instance,substrate aryl groups used in a combinatorial approach can be diverse interms of the core aryl moiety, e.g., a variegation in terms of the ringstructure, and/or can be varied with respect to the other substituents.With respect to the subject invention, for example, it is generallyknown that carboxypeptidases hydrolyze amide bonds. Any biologicallyactive agent having an amine or carboxylic acid moiety may, in theory,be derivatized in a combinatorial approach to form an amide with acarboxylic acid or amine moiety, respectively. For example, a peptidylfragment having a varied number of amino acid residues with a diverseidentity could be coupled to a biologically active agent of interest togive a library of prospective prodrugs, whereupon conversion by reactioncenters such as carboxypeptidases of the prospective prodrugs in thelibrary could be screened. In this fashion, prospective prodrugs of abiologically active agent could be prepared and screened for use with aparticular reaction center, or alternatively, a group of reactioncenters that catalyze a similar type of chemical conversion. As alreadynoted above, the reaction centers themselves may be prepared andscreened by combination methods as well. As will be clear to one ofskill in the art, in preparing any such library, some considerations totake into account include the chemical conversion catalyzed by thereaction center or centers of interest; in what fashion a biologicallyactive agent may be readily derivatized to provide prodrugs so that thereaction center or centers of interest could produce the biologicallyactive agent from a prodrug; and the specificity of the reaction centeror centers of interest to a variation in structure with respect to thereaction that it normally catalyzes, e.g., the naturally occurringsubstrate for an enzyme.

[0254] A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See generallyBlondelle et al. Trends Anal. Chem. 14:83 (1995); U.S. Pat. Nos.5,359,115, 5,362,899, 5,288,514, and 5,721,099; Chen et al. JACS116:2661 (1994; Kerr et al. JACS 115:252 (1993); WO92/10092, WO93/09668,WO 94/08051, WO93/20242 and WO91/07087. Accordingly, a variety oflibraries on the order of about 16 to 1,000,000 or more diversomers canbe synthesized and screened for a particular activity or property.

[0255] In an exemplary embodiment, a library of substituted diversomerscan be synthesized using the subject reactions adapted to the techniquesdescribed in WO 94/08051, e.g., being linked to a polymer bead by ahydrolyzable or photolyzable group, e.g., located at one of thepositions of substrate. According to the technique disclosed therein,the library is synthesized on a set of beads, each bead including a setof tags identifying the particular diversomer on that bead. In oneembodiment, the beads can be dispersed on the surface of a permeablemembrane, and the diversomers released from the beads by lysis of thebead linker. The diversomer from each bead will diffuse across themembrane to an assay zone, where it will interact with an assay for areaction center or centers. Detailed descriptions of a number ofcombinatorial methodologies are provided below.

[0256] (a) Direct Characterization. A growing trend in the field ofcombinatorial chemistry is to exploit the sensitivity of techniques suchas mass spectrometry (MS), e.g., which can be used to characterizesub-femtomolar amounts of a compound, and to directly determine thechemical constitution of a compound selected from a combinatoriallibrary. For instance, where the library is provided on an insolublesupport matrix, discrete populations of compounds can be first releasedfrom the support and characterized by MS. In other embodiments, as partof the MS sample preparation technique, such MS techniques as MALDI canbe used to release a compound from the matrix, particularly where alabile bond is used originally to tether the compound to the matrix. Forinstance, a bead selected from a library can be irradiated in a MALDIstep in order to release the diversomer from the matrix, and ionize thediversomer for MS analysis.

[0257] (b) Multipin Synthesis. The libraries of the subject method cantake the multipin library format. Briefly, Geysen and co-workers, Geysenet al. PNAS 81:3998-4002 (1984), introduced a method for generatingcompound libraries by a parallel synthesis on polyacrylic acid-gratedpolyethylene pins arrayed in the microtitre plate format. The Geysentechnique can be used to synthesize and screen thousands of compoundsper week using the multipin method, and the tethered compounds may bereused in many assays. Appropriate linker moieties can also beenappended to the pins so that the compounds may be cleaved from thesupports after synthesis for assessment of purity and furtherevaluation. Compare Bray et al. Tetrahedron Lett. 31:5811-14 (1990);Valerio et al. Anal Biochem 197:168-77 (1991); Bray et al. TetrahedronLett, 32:6163-66 (1991).

[0258] (c) Divide-Couple-Recombine. In yet another embodiment, avariegated library of compounds can be provided on a set of beadsutilizing the strategy of divide-couple-recombine. See, for example,Houghten PNAS 82:5131-35 (1985); and U.S. Pat. Nos. 4,631,211;5,440,016; 5,480,971. Briefly, as the name implies, at each synthesisstep where degeneracy is introduced into the library, the beads aredivided into separate groups equal to the number of differentsubstituents to be added at a particular position in the library, thedifferent substituents coupled in separate reactions, and the beadsrecombined into one pool for the next iteration.

[0259] In one embodiment, the divide-couple-recombine strategy can becarried out using an analogous approach to the so-called “tea bag”method first developed by Houghten, where compound synthesis occurs onresin sealed inside porous polypropylene bags. Houghten et al. PNAS82:5131-35 (1986). Substituents are coupled to the compound-bearingresins by placing the bags in appropriate reaction solutions, while allcommon steps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

[0260] (d) Combinatorial Libraries by Light-Directed, SpatiallyAddressable Parallel Chemical Synthesis. A scheme of combinatorialsynthesis in which the identity of a compound is given by its locationson a synthesis substrate is termed a spatially-addressable synthesis. Inone embodiment, the combinatorial process is carried out by controllingthe addition of a chemical reagent to specific locations on a solidsupport. Dower et al. Annu Rep Med Chem 26:271-280 (1991); Fodor,Science 251:767 (1991); U.S. Pat. No. 5,143,854; Jacobs et al. TrendsBiotechnol 12:19-26 (1994). The spatial resolution of photolithographyaffords miniaturization. This technique can be carried out through theuse protection/deprotection reactions with photolabile protectinggroups.

[0261] The key points of this technology are illustrated in Gallop etal. J Med Chem 37:1233-51 (1994). A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed. With respect to the above example, for example, alibrary of peptidyl fragments could thereby be prepared, whereupon thebiologically active agent could be coupled in the final step to producea diverse library of prospective prodrugs when paired with reactioncenters that hydrolyze peptide bonds.

[0262] In a light-directed chemical synthesis, the products depend onthe pattern of illumination and on the order of addition of reactants.By varying the lithographic patterns, many different sets of testcompounds can be synthesized simultaneously; this characteristic leadsto the generation of many different masking strategies.

[0263] (e) Encoded Combinatorial Libraries. In yet another embodiment,the subject method utilizes a compound library provided with an encodedtagging system. A recent improvement in the identification of activecompounds from combinatorial libraries employs chemical indexing systemsusing tags that uniquely encode the reaction steps a given bead hasundergone and, by inference, the structure it carries. Conceptually,this approach mimics phage display libraries, where activity derivesfrom expressed peptides, but the structures of the active peptides arededuced from the corresponding genomic DNA sequence. The first encodingof synthetic combinatorial libraries employed DNA as the code. A varietyof other forms of encoding have been reported, including encoding withsequenceable bio-oligomers (e.g., oligonucleotides and peptides), andbinary encoding with additional non-sequenceable tags.

[0264] (1) Tagging with sequenceable bio-oligomers. The principle ofusing oligonucleotides to encode combinatorial synthetic libraries wasdescribed in 1992 Brenner et al. PNAS 89:5381-83 (1992), and an exampleof such a library appeared the following year. Needles et al. PNAS90:10700-04 (1993). A combinatorial library of nominally 7₇ (=823,543)peptides composed of all combinations of Arg, Gln, Phe, Lys, Val, D-Valand TLr (three-letter amino acid code), each of which was encoded by aspecific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), wasprepared by a series of alternating rounds of peptide andoligonucleotide synthesis on solid support. In this work, the aminelinking functionality on the bead was specifically differentiated towardpeptide or oligonucleotide synthesis by simultaneously preincubating thebeads with reagents that generate protected OH groups foroligonucleotide synthesis and protected NH₂ groups for peptide synthesis(here, in a ratio of 1:20). When complete, the tags each consisted of69-mers, 14 units of which carried the code. The bead-bound library wasincubated with a fluorescently labeled antibody, and beads containingbound antibody that fluoresced strongly were harvested byfluorescence-activated cell sorting (FACS). The DNA tags were amplifiedby PCR and sequenced, and the predicted peptides were synthesized.Following such techniques, compound libraries can be derived for use inthe subject method, where the oligonucleotide sequence of the tagidentifies the sequential combinatorial reactions that a particular beadunderwent, and therefore provides the identity of the compound on thebead.

[0265] The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

[0266] Peptides have also been employed as tagging molecules forcombinatorial libraries. Two exemplary approaches are described in theart, both of which employ branched linkers to solid phase upon whichcoding and ligand strands are alternately elaborated. In the firstapproach, Kerr et al. J Am Chem Soc 115:2529-31 (1993), orthogonality insynthesis is achieved by employing acid-labile protection for the codingstrand and base-labile protection for the compound strand.

[0267] In an alternative approach, Nikolaiev et al. Pept Res 6:161-70(1993), branched linkers are employed so that the coding unit and thetest compound can both be attached to the same functional group on theresin. In one embodiment, a cleavable linker can be placed between thebranch point and the bead so that cleavage releases a moleculecontaining both code and the compound. Ptek et al. Tetrahedron Lett32:3891-94 (1991). In another embodiment, the cleavable linker can beplaced so that the test compound can be selectively separated from thebead, leaving the code behind. This last construct is particularlyvaluable because it permits screening of the test compound withoutpotential interference of the coding groups. Examples in the art ofindependent cleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

[0268] (2) Non-sequenceable Tagging: Binary Encoding. An alternativeform of encoding the test compound library employs a set ofnon-sequencable electrophoric tagging molecules that are used as abinary code. Ohlmeyer et al. PNAS 90:10922-26 (1993). Exemplary tags arehaloaromatic alkyl ethers that are detectable as their trimethylsilylethers at less than femtomolar levels by electron capture gaschromatography (ECGC). Variations in the length of the alkyl chain, aswell as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport, Ohlmeyer et al., supra, the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix. Nestler et al. J Org Chem59:4723-24 (1994). This orthogonal attachment strategy permits theselective detachment of library members for assay in solution andsubsequent decoding by ECGC after oxidative detachment of the tag sets.

[0269] Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase, Ohlmeyer et al. PNAS 92:6027-31 (1995), andprovide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

[0270] When prospective prodrugs are screened as libraries of compounds,high throughput assays are desirable in order to maximize the number ofcompounds surveyed in a given period of time. The activity of thereaction center, e.g., enzymatic activity, with any prospective prodrugmay be determined by monitoring either the disappearance of prodrug orthe appearance of the corresponding biologically active agent.Alternatively, the reaction center activity may be determined bymonitoring the reduction or production or any reactants consumed orby-products produced. Spectroscopic methods well-known to those of skillmay be used for such monitoring, or alternatively, any of the reactantsor products, e.g., the prodrug or the corresponding biologically activeagent, may be isolated and quantified.

[0271] In other embodiments, differential assays can be used to identifyprodrugs that react more readily with a encapsulated reaction centerthan with any other naturally occurring enzyme, so that any such prodrugare converted chiefly by any administered encapsulated reaction centerinstead of by any naturally occurring enzymes or catalysts. Such afeature may be desirable depending on how the matrix is used.

[0272] 5.6. Administration

[0273] 5.6.1. Matrix Administration

[0274] Immobilized enzymes may be administered in a variety of ways. Seegenerally Ming et al. Methods for Therapeutic Applications 46:676-699.The site of administration of the matrix may affect its therapeuticeffect depending on the reaction center encapsulated therein. Forexample, the site of implantation of encapsulated PC12 cells fortreatment of Parkinson's disease appears to affect the device output.Emerich et al. Cell Transplant. 5:589-96 (1996).

[0275] A number of different implantation sites in a subject arecontemplated for the matrices of this invention. In particular, the mostpreferred site is determined by the identity of the encapsulatedreaction center. Any site that results in a therapeutic effect may beused. For example, for reaction centers that produce biologically activeagents that are cytotoxic, the implants may be implanted near anyneoplasm. ADEPT technology relies on such proximity to deliver anycytotoxic agent essentially directly to the tumor. In another instance,for matrices used to treat Parkinson's disease by affecting dopaminelevels in the brain, implantation in the brain may be preferred. Othersites in the brain for such matrices include the basal ganglia, thesubstantia nigra, and the striatum.

[0276] The matrices of the present invention may be administered by wayof oral ingestion or implantation. If implantation is desired, they canbe implanted subcutaneously, constitute a part of a prosthesis, or beinserted in a cavity of the human body. Subcutaneous implantation usinga syringe consists of injecting the matrices directly into subcutaneoustissue. Thus, the matrices of the present invention can be suspended ina physiological buffer and introduced via a syringe to the desired site.Other sites include the central nervous system, including the brain,spinal cord, and aqueous and vitreous humors of the eye. Other sites inthe brain include the cerebral cortex, subthalamic nuclei and nucleusBasalis of Maynert. Other sites include the cerebrospinal fluid, thesubarachnoid space, and the lateral ventricles. Other sites includes thekidney subcapsular site, and intraperitoneal and subcutaneous sites.

[0277] In other embodiments of the present invention, the matrices ofthe present invention may be associated with a medical article to beused as an implant. For example, matrices of the present invention couldbe attached as thin films to such devices. Alternatively, matrices ofthe present invention could be attached as a capsule or incorporatedinto any medical device. Exemplary structural medical articles includesuch implants as orthopedic fixation devices, ventricular shunts,laminates for degradable fabric, drug-carriers, burn dressings, coatingsto be placed on other implant devices, and the like.

[0278] For administration of matrices of the present invention, animportant feature may be whether the matrix is intended to stay in placeafter administration or move in the subject. For example, matricesadministered to a subject may be transported and localized in thelymphatic system as part of the subject immune response to the foreignobjects.

[0279] Once a matrix of the present invention is administered, it mayremain in at least partial contact with a biological fluid, such asblood, internal organ secretions, mucus membranes, cerebrospinal fluid,and the like.

[0280] The length of the period during which encapsulated reactioncenter remains active enough so as to produce a therapeutic effect maydepend on a variety of features. Enzymes encapsulated in silica-basedsol-gel matrices have remained active for periods of several months. Theadministration of any matrix of the present invention may result in thelong-term, stable production of a biologically active agent.

[0281] 5.6.2. Formulations and Use of Matrices and Prodrugs

[0282] In addition to the general introduction, pharmaceuticalcompositions for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers or excipients. Thus, as appropriate, matrices andany prodrug, including any physiologically acceptable salts andsolvates, may be formulated for administration by, for example,injection, inhalation or insufflation (either through the mouth or thenose) or oral, buccal, parenteral or rectal administration. Appropriateformulations may depend, in part, on the administration method used andwhether a prodrug or a matrix is being administered.

[0283] The matrices or prodrugs of the invention may be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. Intramuscular, intravenous, intraperitoneal, andsubcutaneous injection is possible. For injection, the matrices orprodrugs of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the prodrugs may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

[0284] For oral administration, the matrices or prodrugs may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0285] Preparations for oral administration may be suitably formulatedto give controlled release of any prodrug. For buccal administration theprodrugs may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the prodrugs foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0286] The prodrugs and/or matrices may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the prodrug may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0287] The prodrugs may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0288] In addition to the formulations described previously, theprodrugs and matrices of the present invention may also be formulated asa depot preparation. Such long acting formulations may be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the prodrugs may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.Other suitable delivery systems include microspheres which offer thepossibility of local noninvasive delivery of drugs over an extendedperiod of time. This technology utilizes microspheres of precapillarysize which can be injected via a coronary catheter into any selectedpart of the e.g. heart or other organs without causing inflammation orischemia. Other methods of controlled release of the prodrugs andmatrices of the present invention are known to those of skill in theart.

[0289] Systemic administration may also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. In addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays or using suppositories. For topical administration, the prodrugsof the invention are formulated into ointments, salves, gels, or creamsas generally known in the art. A wash solution can be used locally totreat an injury or inflammation to accelerate healing.

[0290] The prodrugs and/or matrices may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0291] The prodrugs may be employed in the present invention in variousforms, such as molecular complexes or pharmaceutically acceptable salts.Representative examples of such salts are succinate, hydrochloride,hydrobromide, sulfate, phosphate, nitrate, borate, acetate, maleate,tartrate, salicylate, metal salts (e.g., alkali or alkaline earth),ammonium or amine salts (e.g., quaternary ammonium) and the like.Furthermore, derivatives of the prodrugs such as esters, amides, andethers which have desirable retention and release characteristics butwhich are readily hydrolyzed in vivo by physiological pH or enzymes canalso be employed.

[0292] 5.7. Treatment

[0293] The selected dosage level for the matrices and prodrugs, ifapplicable, of the present invention will depend upon a variety offactors including: the load of the reaction center within the matrix;the activity of the reaction center, the activity of the particularprodrug employed, or the ester, salt or amide thereof; the route ofadministration; the time of administration, the rate of excretion of theparticular prodrug (and possibly matrix) being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular matrix and prodrug employed, the age,sex, weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

[0294] Toxicity and therapeutic efficacy of the matrices of the presentinvention may be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. In certain embodiments, thosein which an exogenous prodrug is activated by the reaction centerencapsulated in a biocompatible matrix of the subject invention, theefficacy of treatment using the subject invention may be gauged bycomparing any of the foregoing parameters resulting from treatment usinga prodrug alone (i.e., without the subject matrix), the biologicallyactive substance that results from the prodrug alone, and treatmentusing the prodrug and a subject matrix as disclosed herein. In certainembodiments, treatments using the subject invention have ratios of abouttwo (or less), five, ten, one hundred, one thousand or even greaterorders of magnitude more favorable than treatment with the prodrug aloneor the biologically active substance that results from the prodrugalone.

[0295] Because the matrix itself does not result in a therapeutic effectwithout the involvement of some other compound, e.g., a prodrug ornaturally occurring metabolite, but also the availability of othercompounds that interact with the matrix may affect any treatment regime.In general, matrices and the biologically active agents that theyproduce which exhibit large therapeutic indices are preferred. Bytargeting the matrix to a particular region of a subject so as tolocalize the production of the biologically active agent, thetherapeutic efficacy may be dramatically increased, and unwanted sideeffects may be minimized. For example, by implanting the dopamineproducing matrix in the striatum, it may not be necessary to administerL-dopa with carbidopa or benserazide, which is used to combat nausearesulting from conversion of L-dopa to dopamine outside of the brain.

[0296] Because the matrix, upon administration, may be in place andactive for significant time periods, ant treatment regime may involvemultiple administrations of a prodrug so as to produce biologicallyactive agent.

[0297] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any matrix and/orprodrug used in the present invention, the therapeutically effectivedose can be estimated initially from cell culture assays. For example, adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma of abiologically active agent may be measured, for example, by highperformance liquid chromatography.

Exemplifications

[0298] The present invention now being generally described, it may bemore readily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention in any way.

[0299] A. Reaction Center Encapsulation Studies

[0300] 1. Matrix Preparation

[0301] The general synthetic technique used for preparation of thesilica sol was addition of 21 mL of tetramethyl orthosilicate (Aldrich,99+%) and 5.08 mL of a 4 mM HCl solution to a 25×150 mm test tubeequipped with a stirbar. The mixture is stirred until homogeneous(approximately 15 minutes). The test tube containing the sol is thentransferred to an ice bath and allowed to cool for 10 minutes. A 2 mLaliquot of sol is then transferred to another chilled test tube in anice bath and stirred. To this sol, 1 mL of chilled buffer solution(appropriate to the enzyme to be entrapped) is added, and stirred forca. 10 s, followed by addition of 1 mL of chilled, buffered solutioncontaining the desired enzyme. The sol is swirled briefly, and thenpipetted into a 4.5 mL polystyrene cuvette (cell culture dishes werealso used for surface area study matrices). The cuvette opening issealed with Parafilm following gel formation (cell culture dish coverswere used for surface area study matrices). The gel is then allowed toage in the sealed container for a period of time ranging from 18 h to 50d or more at temperatures ranging from 4° C. to room temperature.Selected samples were dried at ambient temperature over a period of daysto weeks by puncturing the Parafilm covering the container opening.Other samples were assayed without drying.

[0302] 2. Enzyme Encapsulation and Assays

[0303] a) b-Glucosidase

[0304] The entrapment of b-Glucosidase (from almonds, crude, lyophilizedpowder, Sigma) was performed as outlined above, using 50 mM, pH 5.0acetate buffer. The activity assay was performed using 2.667 mL of a 10mM solution of para-nitrophenyl-b-D-glucopyranoside (Sigma, 99+%) inbuffer and 37.33 mL acetate buffer in a 125 mL Erlenmeyer flask (40 mLtotal solution volume). 2 mL aliquots of solution were removed for assayand their UV-Vis spectra recorded.

[0305] b) Penicillinase

[0306] Entrapment of Penicillinase (Type I from Bacillus cereus,lyophilized powder containing approx. 10% protein, Sigma) was performedas outlined above, using 50 mM pH 6.5 phosphate buffer. Penicillinaseactivity was determined using 100 mL of a 3 mM solution of Penicillin G(Benzylpenicillin, sodium salt, Sigma) in buffer. 2 mL aliquots of thereaction solution were removed for assay and their UV-Vis spectrarecorded.

[0307] c) Tyrosinase

[0308] Entrapment of Tyrosinase (from mushroom, Sigma) was performed asoutlined above, with 50 mM pH 6.5 phosphate buffer solution. Tyrosinaseactivity assays were performed using 0.3 mM L-tyrosine (Aldrich; 99+%)solution in buffer.

[0309] d) Tyrosine Decarboxylase

[0310] Entrapment of Tyrosine Decarboxylase (from Streptococcusfaecalis, Fluka) was performed using 50 mM pH 5.5 acetate buffer and wasaccomplished by the method outlined above. The activity assay wasaccomplished using a 50:50 mixture of 2.5 mM solution of L-tyrosine(Aldrich, 99+%) in buffer and buffer. Total reaction volume for thisassay was either 40 mL (19.5 h data reported herein) or 100 mL (allother data reported). 2 mL aliquots of reaction mixture were removedfrom the reaction vessel for assay. The method used for this assay wasaddition of 1 mL of a 1M K2CO3 solution to the 2 mL aliquot, followed bymixing. To this was added 2 drops of a solution of picrylsulfonic acid(5% w/v aquesous solution of 2,4,6-trinitrobenzenesulfonic acid, Sigma).The mixture was mixed well. 2 mL of toluene were added to this mixture,the layers shaken well, and centrifuged. The toluene layer was removedand its UV-Vis spectrum collected. Where applicable, a 0.1 mM solutionof pyridoxal-5-phosphate monohydrate (98%, Aldrich) in buffer wassubstituted for the buffer solution in the assay mixture. Assaysperformed in the presence of cofactor were carried out in foil-coveredreaction vessels due to the sensitivity of pyridoxal-5-phosphate tolight.

[0311] 3. Results of Enzyme Encapsulation and Assays

[0312] a) b-Glucosidase entrapment yielded active matrices which wereassayed using the synthetic substratepara-nitrophenyl-b-D-glucopyranoside, shown below. Enzymatic activity ofmatrix composites on the synthetic substrate results in cleavage of theglucosidic bond producing a bathochromic shift in the spectral band.This shift permits monitoring of the cleavage process, as illustrated inFIG. 1.

[0313] The synthetic substrate para-nitrophenyl-b-D-glucopyranoside.

[0314] b) Penicillinase entrapment provided active matrices which wereassayed using the sodium salt of the synthetic substratebenzylpenicillin, shown below. Conversion of penicillin to penicilloicacid via rupture of the β-lactam ring may be monitoredspectrophotometrically, as shown in FIG. 2.

[0315] Substrate used for penicillinase activity assay, sodiumbenzylpenicillin.

[0316] Reproducibility of the measurements done for penicillinase waschecked by performing activity assay multiple times for the same matrix.As shown in FIG. 3(a), good agreement is observed for multiple assaysperformed over six consecutive days. Likewise, running multiple matricesfrom the same preparation to check reproducibility of the matrixentrapment shows good agreement, as seen in FIG. 3(b).

[0317] Loading studies utilizing penicillinase matrices were performedin which the enzyme concentration was varied over a wide range todtermine the optimal enzyme concentration. The bar graph shown in FIG. 4shows the effects of varying enzyme concentration on the activity of thematrix. The highest percentage of activity observed as a function ofenzyme entrapped within the matrix (selected from the five compositionsanalyzed) occurs for the lowest concentration of enzyme examined, asshown in FIG. 5.

[0318] Surface area effects were also examined utilizing penicillinasematrices. Ten identical monoliths were prepared and aged simultaneously.Five of these were assayed as whole monoliths (cast in 4.5 mL cuvettes)while another five matrices were coarsely crushed and then assayed. Thisqualitative examination of surface area effects revealed that anincrease in surface area does result in an increase in the enzymeactivity observed, as shown in FIGS. 6 and 7. It should be noted thatthere is no leaching of enzyme observed from either the whole or crushedmatrices, as determined by soaking the matrices in buffer solutionovernight and subsequently checking the activity of the soak solution.The reproducibility of the measurements for the assays shown in FIG. 6is quite reasonable, with the larger deviation in the crushed matricesattributable to the lack of control over particle size when breaking upthe samples. FIG. 7, showing the mean values for each measurement witherror bars, emphasizes the greater relative activity of the crushedmatrix samples.

[0319] The significant effect of changing surface area on the observedenzyme activity prompted further investigation. Control over totalsurface area was achieved by casting the sol containing penicillinaseinto varying numbers of cell culture plate wells (22.6 mm diameter). Byvarying the amount of sol cast into a given well, the total 4 mL ofmaterial per matrix could be spread out over a number of wells and thedisks cast in these wells could be recombined, after removal from thewells, for assay. Thus, the 4 mL of sol that constitutes one matrixpreparation could be cast into one or multiple wells to generate sampleswith known, varying surface areas. Surface area stated for a givenmatrix reflects the initial surface area of the gel when freshly cast,and does not attempt to correct for any shrinkage that occurred duringaging. FIG. 8(a) illustrates the difference in activity observed formatrices of varying surface areas. FIG. 8(b) shows the activity as apercentage of the penicillinase activity used in the preparation of thematrices.

[0320] c) Following entrapment of tyrosinase, the bifunctional activityof this enzyme was found to complicate spectrophotometric assay of thematrix composite due to the variation in molar extinction coefficient ofthe different species, and possible retention within the matrix.Tyrosinase possesses both cresolase (conversion of phenols to diphenols)and catecholase activity (conversion of diphenols to the correspondingquinone), as shown below. However, a qualitative analysis shows theconversion of the natural substrate L-tyrosine to L-dopa, which thenundergoes dehydrogenation to give dopaquinone. Dopaquinone is unstablein aqueous solutions and undergoes a Michealis rearrangement to form,among other products, a number of melanin precursors which eventuallypolymerize to produce pigments. This complicated reaction may befollowed qualitatively by monitoring a color change within the matrix.Although the L-tyrosine solution is, itself, colorless, thetyrosinase-containing matrices become noticeably darkened within onehour of contact with the substrate solution, suggesting formation ofproducts with subsequent retention by the matrix. A solution of L-dopain the presence of tyrosinase, likewise forms a gray-black precipitate.

[0321] d) Active Tyrosine Decarboxylase matrices were and assayed usingL-tyrosine as substrate. A complicating factor in the assay of entrappedTyrosine Decarboxylase was the unavailability of a directspectrophotometric method due to the equivalent molar extinctioncoefficients of substrate and product. The observation necessitated thedevelopment of an indirect assay provided by Phan et. al., App. Biochem.Biotech. 8:127 (1983). The results of an active Tyrosine Decarboxylasecomposite assay are shown in FIG. 9.

[0322] Longer aging times for Tyrosine Decarboxylase-containing matricesresulted in matrices for which no enzyme activity was observed withoutaddition of cofactor. Addition of cofactor, pyridoxal-5-phosphatemonohydrate (0.05 mM), to the assay mixture restored activity of theentrapped enzyme to varying degrees depending on the aging of themonolith. FIG. 10 shows activity assays for two 16 day old TyrosineDecarboxylase-containing matrices, one without cofactor present and onewith cofactor, and compares them to a matrix of the same compositionassayed after aging 19 h. The activity observed at 19 h without cofactorand at 16 d with cofactor present are nearly identical, whereas withoutthe presence of cofactor little, if any, activity is noted. For matricesaged 50 d a significant portion of the activity is retained in thepresence in cofactor, although some loss of activity is observed.

[0323] B. Matrix Optimization Studies

[0324] 1. Matrix Preparation

[0325] The general synthetic technique used for preparation of thesilica sol was addition of appropriate aliquots of the organicallysubstituted trimethoxysilane, tetramethyl orthosilicate and 4 mM HClsolution to a 25×150 mm test tube equipped with a stirbar. Total desiredvolume of sol was determined by the number of matrices to be prepared.The RSi(OCH₃)₃ and TMOS reagents were combined in appropriate ratios toyield the desired compositions. Reagent Source Tetramethylorthosilicate(TMOS) Aldrich, 99+% Methyltrimethoxysilane (MTMS) Aldrich, 98%Ethyltrimethoxysilane (ETMS) Aldrich, 97+% Trimethoxypropylsilane (TMPS)Aldrich, 98% iso-Butyltrimethoxysilane (i-BTMS) Aldrich, 97%n-Butyltrimethoxysilane (n-BTMS) United Chemical Technologies, 95.3%Phenyltrimethoxysilane (PTMS) Aldrich, 97%

[0326] As with 100% TMOS matrices, the mixture is stirred untilhomogeneous (approximately 15 minutes). The test tube containing the solis then transferred to an ice bath and allowed to cool for 10 minutes. A2 mL aliquot of sol is then transferred to another chilled test tube inan ice bath and stirred. To this sol, 1 mL of chilled buffer solution(appropriate to the enzyme to be entrapped) is added, and stirred forca. 10 s, followed by addition of 1 mL of chilled, buffered solutioncontaining the desired enzyme. The sol is swirled briefly, and thenpipetted into a 4.5 mL polystyrene cuvette (cell culture dishes werealso used for surface area study matrices). The cuvette opening issealed with Parafilm following gel formation (cell culture dish coverswere used for surface area study matrices). The gel is then allowed toage in the sealed container for a period of time ranging from 14 to 50days or more at temperatures ranging from 4° C. to room temperature.

[0327] 2. Enzyme Encapsulation and Assays

[0328] Entrapment of Penicillinase (Type 1 from Bacillus cereus,lyophillized powder containing approx. 10% protein, Sigma) was performedas outlined above, using 50 mM pH 6.5 phosphate buffer. Penicillinaseactivity was determined using 100 mL of a 3 mM solution of Penicillin G(Benzylpenicillin, sodium salt, Sigma) in buffer. 2 mL aliquots of thereaction solution were removed for assay and their UV-Vis spectrarecorded.

[0329] 3. Results of Enzyme Encapsulation and Assays

[0330] Initial examination of which matrix compositions providedmatrices suitable for the purposes of this study excluded then-butyltrimethoxysilane composition, as well as some of the higherratios of other RSi(OCH₃)₃ precursors, due to the failure of thesecompositions to form a gel that was appropriate for our intended uses.Compositions examined, and their reactivity relative to 100% TMOSmatrices are shown in Table 1. TABLE 1 Activity for given compositionsrelative to 100% TMOS. Composition Relative Activity MTMS:TMOS 10%MTMS:90% TMOS 108%  20% MTMS:80% TMOS 82% 30% MTMS:70% TMOS 92% 40%MTMS:60% TMOS 104%  50% MTMS:50% TMOS 112%* ETMS:TMOS 10% ETMS:90% TMOS99% 20% ETMS:80% TMOS 93% 30% ETMS:70% TMOS 99% TMPS:TMOS 10% TMPS:90%TMOS 100%  20% TMPS:80% TMOS 80% i-BTMS:TMOS 10% i-BTMS:90% TMOs 90% 20%i-BTMS:80% TMOs 75% PTMS:TMOS 10% PTMS:90% TMOS 94%

[0331] In addition, it was observed that as the matrices age therelative activity of the MTMS-containing matrices with respect to 100%TMOS drops. When matrices from the same preparation are assayed afteraging 104 days at 4° C., the relative activity observed is shown inTable 2. TABLE 2 Enzyme activity relative to 100% TMOS for varyingMTMS-containing matrices aged 104 days. Composition Relative ActivityMTMS:TMOS 10% MTMS:90% TMOS 85% 20% MTMS:80% TMOS 78% 30% MTMS:70% TMOS85% 40% MTMS:60% TMOS 85% 50% MTMS:50% TMOS 103%*

[0332] All publications and patents mentioned herein are herebyincorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference and set forth in its entirety herein. In caseof conflict, the present application, including any definitions herein,will control. In addition to the foregoing materials, the practice ofthe present invention may employ in part, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning aLaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatiseMethods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods in Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook ofExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986), all of whichreferences are hereby incorporated by reference to the same extent asthe other references specified herein.

[0333] The specification and examples should be considered exemplaryonly with the true scope and spirt of the invention suggested by thefollowing claims.

What is claimed is:
 1. A method for producing a biologically activeagent from a prodrug, comprising: a. encapsulating a first cell-freereaction center in a biocompatible matrix; and b. administering saidbiocompatible matrix to a subject; wherein said biocompatible matrixcomprises an inorganic-based sol-gel matrix and wherein said firstreaction center converts a first prodrug into a first biologicallyactive agent in said subject.
 2. The method of claim 1, wherein saidbiocompatible matrix comprises a silica-based sol-gel matrix.
 3. Themethod of claim 2, wherein said first reaction center comprises one ofthe following: an enzyme, an antibody or a catalytic antibody.
 4. Themethod of claim 2, wherein said biocompatible matrix encapsulates atleast one additive.
 5. The method of claim 2, wherein said firstreaction center comprises L-amino acid decarboxylase.
 6. The method ofclaim 5, wherein said first prodrug comprises L-dopa and said firstbiologically active agent comprises dopamine.
 7. The method of claim 2,wherein said first reaction center comprises L-tyrosine decarboxylase.8. The method of claim 7, wherein said first prodrug comprises L-dopaand said first biologically active agent comprises dopamine.
 9. Themethod of claim 2, further comprising encapsulating a second reactioncenter in said biocompatible matrix before administering saidbiocompatible matrix to said subject.
 10. The method of claim 9, whereinsaid first biologically active agent produced by said first reactioncenter from said first prodrug is a second prodrug for said secondreaction center, and wherein said second reaction center produces asecond biologically active agent that differs from said firstbiologically active agent.
 11. The method of claim 12, wherein saidfirst reaction center comprises tyrosine monooxygenase, and said secondreaction center is one of the following: L-amino acid decarboxylase orL-tyrosine decarboxylase.
 12. The method of claim 11, wherein said firstprodrug comprises tyrosine, said first biologically active agent andsaid second prodrug comprises L-dopa, and said second biologicallyactive agent comprises dopamine.
 13. The method of claims 4, 5, 6, 7, 8,11 or 12, wherein administering said biocompatible matrix comprisesadministering said biocompatible matrix to a region of the brain of saidsubject.
 14. The method of claim 13, wherein said region of said brainof said subject is one of the following: basal ganglia, substantia nigraor striatum.
 15. The method of claim 2, wherein said biocompatiblematrix is prepared from at least one type of oxysilane.
 16. The methodof claim 15, wherein said biocompatible matrix is prepared from morethan one type of oxysilane.
 17. The method of claim 15, wherein saidbiocompatible matrix is prepared from at least one type of inorganicoxide and at least one type of oxysilane.
 18. The method of claim 15 or16, wherein said type of oxysilane has at least one non-hydrolizablesubstituent.
 19. The method of claim 2, wherein said biocompatiblematrix consists essentially of siloxane.
 20. The method of claim 2,wherein said biocompatible matrix comprises siloxane.
 21. The method ofclaim 2, wherein administering said biocompatible matrix comprisessurgical implantation.
 22. The method of claim 2, further comprisingadministering said first prodrug to said subject.
 23. The method ofclaim 2, wherein said first prodrug comprises an exogenous prodrug. 24.The method of claim 2, wherein said first prodrug comprises anendogenous prodrug.
 25. The method of claim 2, wherein said firstreaction center comprises an enzyme or antibody that is xenogeneic tosaid subject.
 26. The method of claim 3, wherein the ratio of Km(nonencapsulated) to Km (encapsulated) for said first reaction center isgreater than or equal to one.
 27. The method of claim 3, wherein theratio of Km (nonencapsulated) to Km (encapsulated) for said firstreaction center is less than or equal to one.
 28. The method of claim 2,wherein said first reaction center comprises more than one weightpercent of said biocompatible matrix.
 29. The method of claim 2, whereinsaid first reaction center comprises less than one weight percent ofsaid biocompatible matrix.
 30. The method of claim 29, wherein saidfirst reaction center comprises more than five weight percent of saidbiocompatible matrix.
 31. The method of claim 31, wherein said firstreaction center comprises more than ten weight percent of saidbiocompatible matrix.
 32. The method of claim 2, wherein said firstreaction center is attached to said biocompatible matrix.
 33. The methodof claim 2, wherein said biocompatible matrix is immunoisolatory. 34.The method of claim 2, wherein administering said biocompatible matrixcomprises parenteral administration.
 35. The method of claim 2, whereinadministering said biocompatible matrix comprises systemicadministration.
 36. The method of claim 2, wherein treatment of saidsubject by said method results in long-term, stable production of saidfirst biologically active agent in said subject.
 37. The method of claim22, wherein said first prodrug is administered to said subject on atleast more than one occasion.
 38. The method of claim 2, wherein saidfirst biologically active agent is cytotoxic.
 39. The method of claim38, wherein said biocompatible matrix is implanted in proximity to aneoplasm.
 40. The method of claim 2, wherein said first reaction centerdoes not leach significantly from said biocompatible matrix.
 41. Themethod of claim 2, wherein said biocompatible matrix comprises axero-gel.
 42. The method of claim 15, wherein said oxysilane is one ofthe following: TMOS or TEOS.
 43. The method of claim 3, wherein saidbiocompatible matrix causes prodrug activation.
 44. The method of claim2, wherein said first prodrug is a deleterious agent to said subject andsaid first biologically active agent is less deleterious to said subjectthan said first prodrug.
 45. The method of claim 44, wherein said firstprodrug is an agent to which said subject is capable of becomingaddicted, and wherein said subject is less capable of becoming addictedto said first biologically active agent.
 46. The method of claim 45,wherein said first prodrug is one of the following: ethanol or cocaine.47. The method of claim 2, wherein said first prodrug is one of thefollowing: L-phenylalanine, noradrenalin, norepinephrine, histadine,histamine, 1-methylhistamine, glutumate, GABA or serine.
 48. The methodof claim 2, wherein said subject is human.
 49. The method of claim 2,wherein said subject receives a therapeutically effective amount of saidbiocompatible matrix and said first prodrug.
 50. The method of claim 23,wherein the ratio of the therapeutic index of treatment using said firstprodrug and said biocompatible matrix over the therapeutic index oftreatment using said first prodrug alone is about five or more.
 51. Themethod of claim 50, wherein the ratio of the therapeutic index oftreatment using said first prodrug and said biocompatible matrix overthe therapeutic index of treatment using said first prodrug alone isabout ten or more.
 52. The method of claim 51, wherein the ratio of thetherapeutic index of treatment using said first prodrug and saidbiocompatible matrix over the therapeutic index of treatment using saidfirst prodrug alone is at least about one hundred.
 53. The method ofclaim 37, wherein the ratio of the therapeutic index of treatment usingsaid first prodrug and said biocompatible matrix over the therapeuticindex of treatment using the biologically active agent of said firstprodrug alone is at about five or more.
 54. The method of claim 53,wherein the ratio of the therapeutic index of treatment using said firstprodrug and said biocompatible matrix over the therapeutic index oftreatment using the biologically active agent of said first prodrugalone is at about ten or more.
 55. The method of claim 51, wherein theratio of the therapeutic index of treatment using said first prodrug andsaid biocompatible matrix over the therapeutic index of treatment usingthe biologically active agent of said first prodrug alone is at leastabout one hundred.
 56. The method of claim 2, wherein said firstbiologically active agent comprises a neutrophic factor.
 57. The methodof claim 2, wherein said first biologically active agent comprises atype selected from the group consisting of anti-angiogenesis factors,antiinfectives; antibiotics agents; antiviral agents; analgesics;anorexics; antihelmintics; antiarthritics; antiasthmatic agents;anticonvulsants; antidepressants; antidiuretic agents; antidiarrheals;antihistamines; antiinflammatory agents; antimigraine preparations;antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics;antipsychotics; antipyretics, antispasmodics; anticholinergics;sympathomimetics; xanthine derivatives; cardiovascular preparations;calcium channel blockers; beta-blockers; antiarrhythmics;antihypertensives; catecholamines; diuretics; vasodilators; centralnervous system stimulants; cough preparations; cold preparations;decongestants; growth factors, hormones; steroids,; corticosteroids;hypnotics; immunosuppressives; muscle relaxants; parasympatholytics;psychostimulants; sedatives; tranquilizers; proteins; polysaccharides;glycoproteins; lipoproteins; interferons; cytokines; chemotherapeuticagents; anti-neoplastics, antibiotics, anti-virals, anti-fungals,anti-inflammatories, anticoagulants, lymphokines, and antigenicmaterials.
 58. The method of claim 3, wherein said first reaction centercomprises an enzyme that is a member of a class selected from the groupconsisting of oxidoreductases; transferases; hydrolases; isomerases; andligases.
 59. The method of claim 2, wherein said first reaction centerreplaces, augments or supplements some endogenous biological activity insaid subject.
 60. The method of claim 59, wherein said first reactioncenter comprises an enzyme in which said subject is deficient.
 61. Themethod of claim 60, wherein said first reaction center is one of thefollowing: glucocerebrosidase; α-1,4-glucosidase; α-galactosidase;α-L-iduronidase; β-glucuronidase; aminolaevulinate dehydratase;bilirubin oxidase; catalase; fibrinolysin; glutaminase; hemoglobin;heparinase; L-arginine ureahydrolase (A1); arginase; liver microsomalenzymes; phenylalanine ammonia lyase; streptokinase; superoxidedismutase; terrilythin; tyrosinase; UDP glucuronyl transferase; ureacycle enzymes; urease; uricase; or urokinase.
 62. A method of toxicologytesting, comprising: a. encapsulating at least one reaction center in asilica-based sol-gel matrix; b. interacting a compound with said matrix;and, c. evaluating for any products of said compound resulting fromconversion of said compound by said reaction center, wherein productionof any cytotoxic or mutagenic products indicates that said compound maybe toxic to a subject upon administration.
 63. The method of claim 62,wherein said reaction center comprises an enzyme that is located in theliver of a mammal.
 64. The method of claim 62, wherein said reactioncenter is an enzyme prepared by recombinant methods.
 65. The method ofclaim 62, wherein said reaction center is cell-free.
 66. The method ofclaim 63, wherein said mammal is one of the following: a pig or a human.67. A biocompatible matrix for treatment, comprising: a. ainorganic-based sol-gel matrix that is biocompatible; and, b. a firstcell-free reaction center encapsulated in said matrix, wherein saidfirst reaction center, after administration of said matrix to a subject,produces a therapeutically effective amount of a first biologicallyactive agent from a first prodrug in said subject.
 68. The biocompatiblematrix of claim 67, wherein said biocompatible matrix comprises asilica-based sol-gel matrix.
 69. The biocompatible matrix of claim 67,wherein said first reaction center comprises one of the following: anenzyme, an antibody or a catalytic antibody.
 70. The biocompatiblematrix of claim 68, wherein said first reaction center comprises one ofthe following: L-amino acid decarboxylase or L-tyrosine decarboxylase.71. The biocompatible matrix of claim 71, wherein said first reactioncenter comprises L-amino acid decarboxylase, said first prodrugcomprises L-dopa, and said first biologically active agent comprisesdopamine.
 72. The biocompatible matrix of claim 68, wherein saidbiocompatible matrix further comprises a second reaction center.
 73. Thebiocompatible matrix of claim 72, wherein said first biologically activeagent produced by said first reaction center from said first prodrug isa second prodrug for said second reaction center, and wherein saidsecond reaction center produces a second biologically active agent thatdiffers from said first biologically active agent.
 74. The biocompatiblematrix of claim 73, wherein said first reaction center comprisestyrosine monooxygenase, and said second reaction center is one of thefollowing: L-amino acid decarboxylase or L-tyrosine decarboxylase. 75.The biocompatible matrix of claims 70, 71 or 74, wherein administeringsaid biocompatible matrix comprises administering said biocompatiblematrix to a region of the brain of said subject.
 76. The biocompatiblematrix of claim 75, wherein said region of said brain of said subject isone of the following: basal ganglia, substantia nigra or striatum. 77.The biocompatible matrix of claim 68, wherein said biocompatible matrixis prepared from at least one type of oxysilane.
 78. The biocompatiblematrix of claim 68, wherein said biocompatible matrix is siloxane. 79.The biocompatible matrix of claim 77, wherein said type of oxysilane hasat least one non-hydrolizable substituent.
 80. The biocompatible matrixof claim 68, wherein said first prodrug is exogenous to said subject.81. The biocompatible matrix of claim 67, wherein said first prodrug isendogenous to said subject.
 82. The biocompatible matrix of claim 68,wherein said first reaction center comprises an enzyme that isxenogeneic to said subject.
 83. The biocompatible matrix of claim 69,wherein the ratio of Km (nonencapsulated) to Km (encapsulated) for saidfirst reaction center is greater than or equal to one.
 84. Thebiocompatible matrix of claim 67, wherein said first reaction centercomprises more than one weight percent of said biocompatible matrix. 85.The biocompatible matrix of claim 82, wherein said xenogeneic enzymecomprises more than five weight percent of said biocompatible matrix.86. The biocompatible matrix of claim 68, wherein said first reactioncenter comprises more than ten weight percent of said biocompatiblematrix.
 87. The biocompatible matrix of claim 68, wherein saidbiocompatible matrix is immunoisolatory.
 88. The biocompatible matrix ofclaim 68, wherein said biocompatible matrix is capable of long-term,stable production of said first biologically active agent in saidsubject.
 89. The biocompatible matrix of claim 69, wherein said firstbiologically active agent is cytotoxic.
 90. The biocompatible matrix ofclaim 67, wherein said first reaction center does not leachsignificantly from said biocompatible matrix after administration. 91.The biocompatible matrix of claim 68, wherein said biocompatible matrixcomprises a xero-gel.
 92. The biocompatible matrix of claim 77, whereinsaid oxysilane is one of the following: TMOS or TEOS.
 93. Thebiocompatible matrix of claim 68, wherein said first prodrug is adeleterious agent to said subject and said first biologically activeagent is less deleterious to said subject than said first prodrug. 94.The biocompatible matrix of claim 67, wherein said first prodrug is anagent to which said subject is capable of becoming addicted, and whereinsaid subject is less capable of becoming addicted to said firstbiologically active agent.
 95. The biocompatible matrix of claim 68,wherein said first prodrug is one of the following: L-phenylalanine,noradrenalin, norepinephrine, histadine, histamine, 1-methylhistamine,glutamate, GABA or serine.
 96. The biocompatible matrix of claim 80,wherein the ratio of the therapeutic index of treatment using said firstprodrug and said first biocompatible matrix over the therapeutic indexof treatment using said first prodrug alone is about five or more. 97.The biocompatible matrix of claim 88, wherein the ratio of thetherapeutic index of treatment using said first prodrug and said firstbiocompatible matrix over the therapeutic index of treatment using saidfirst prodrug alone is at least about one hundred.
 98. The biocompatiblematrix of claim 80, wherein the ratio of the therapeutic index oftreatment using said first prodrug and said biocompatible matrix overthe therapeutic index of treatment using the biologically active agentof said first prodrug alone is at least about ten or more.
 99. Thebiocompatible matrix of claim 80, wherein said first biologically activeagent comprises a type selected from the group consisting ofanti-angiogenesis factors, antinfectives; antibiotics agents; antiviralagents; analgesics; anorexics; antihelmintics; antiarthritics;antiasthmatic agents; anticonvulsants; antidepressants; antidiureticagents; antidiarrheals; antihistamines; antiinflammatory agents;antimigraine preparations; antinauseants; antineoplastics;antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics,antispasmodics; anticholinergics; sympathomimetics; xanthinederivatives; cardiovascular preparations; calcium channel blockers;beta-blockers; antiarrhythmics; antihypertensives; catecholamines;diuretics; vasodilators; central nervous system stimulants; coughpreparations; cold preparations; decongestants; growth factors,hormones; steroids,; corticosteroids; hypnotics; immunosuppressives;muscle relaxants; parasympatholytics; psychostimulants; sedatives;tranquilizers; proteins; polysaccharides; glycoproteins; lipoproteins;interferons; cytokines; chemotherapeutic agents; anti-neoplastics,antibiotics, anti-virals, anti-fungals, anti-inflammatories,anticoagulants, lymphokines, and antigenic materials.
 100. Thebiocompatible matrix of claim 68, wherein said first reaction centercomprises an enzyme that is member of a class of one of the following:oxidoreductases; transferases; hydrolases; isomerases; or ligases. 101.The biocompatible matrix of claim 67, wherein said first reaction centerreplaces, augments or supplements some endogenous biological activity insaid subject.
 102. The biocompatible matrix of claim 68, wherein saidfirst reaction center comprises an enzyme in which said subject isdeficient.
 103. The biocompatible matrix of claim 102, wherein saidfirst reaction center is one of the following: glucocerebrosidase;α-1,4-glucosidase; α-galactosidase; α-L-iduronidase; β-glucuronidase;aminolaevulinate dehydratase; bilirubin oxidase; catalase; fibrinolysin;glutaminase; hemoglobin; heparinase; L-arginine ureahydrolase (A1);arginase; liver microsomal enzymes; phenylalanine ammonia lyase;streptokinase; superoxide dismutase; terrilythin; tyrosinase; UDPglucuronyl transferase; urea cycle enzymes; urease; uricase; orurokinase.
 104. A biologically active agent produced by a processcomprising: a. encapsulating a first cell-free reaction center in abiocompatible matrix; and b. administering said biocompatible matrix toa subject; wherein said biocompatible matrix comprises aninorganic-based sol-gel matrix, and wherein said biologically activeagent is produced by said first reaction center from a first prodrug insaid subject.
 105. The biologically active agent of claim 104, whereinsaid biocompatible matrix comprises a silica-based sol-gel matrix. 106.The biologically active agent of claim 105, wherein said first reactioncenter comprises one of the following: an enzyme, an antibody or acatalytic antibody.
 107. The biologically active agent of claim 106,wherein said first reaction center is one of the following: L-amino aciddecarboxylase, L-tyrosine decarboxylase or tyrosine monooxygenase, 108.The biologically active agent of claim 105, further comprisingencapsulating a second reaction center in said biocompatible matrixbefore administering said biocompatible matrix to said subject.
 109. Thebiologically active agent of claim 107, wherein administering saidbiocompatible matrix comprises administering said biocompatible matrixto one of the following regions of the brain: basal ganglia, substantianigra or striatum.
 110. The biologically active agent of claim 105,further comprising preparing said biocompatible matrix from at least onetype of oxysilane at substantially the same time as said encapsulatingof said first reaction center.
 111. The biologically active agent ofclaim 105, wherein said biocompatible matrix consists essentially ofsiloxane.
 112. The biologically active agent of claim 105, whereinadministering said biocompatible matrix to a subject comprises surgicalimplantation.
 113. The biologically active agent of claim 110, furthercomprising administering said first prodrug to said subject.
 114. Thebiologically active agent of claim 105, wherein said first prodrugcomprises a prodrug exogenus to said subject.
 115. The biologicallyactive agent of claim 105, wherein said process results in long-term,stable production of said biologically active agent in said subject.116. The biologically active agent of claim 114, wherein said firstprodrug is administered to said subject on at least more than oneoccasion.
 117. The biologically active agent of claim 105, wherein saidfirst prodrug is a deleterious agent to said subject and said firstbiologically active agent is less deleterious to said subject than saidfirst prodrug.
 118. The biologically active agent of claim 105, whereinsaid subject is human.
 119. The biologically active agent of claim 105,wherein said biologically active agent comprises a type selected fromthe group consisting of anti-angiogenesis factors, antiinfectives;antibiotics agents; antiviral agents; analgesics; anorexics;antihelmintics; antiarthritics; antiasthmatic agents; anticonvulsants;antidepressants; antidiuretic agents; antidiarrheals; antihistamines;antiinflammatory agents; antimigraine preparations; antinauseants;antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics, antispasmodics; anticholinergics; sympathomimetics;xanthine derivatives; cardiovascular preparations; calcium channelblockers; beta-blockers; antiarrhythmics; antihypertensives;catecholamines; diuretics; vasodilators; central nervous systemstimulants; cough preparations; cold preparations; decongestants; growthfactors, hormones; steroids,; corticosteroids; hypnotics;immunosuppressives; muscle relaxants; parasympatholytics;psychostimulants; sedatives; tranquilizers; proteins; polysaccharides;glycoproteins; lipoproteins; interferons; cytokines; chemotherapeuticagents; anti-neoplastics, antibiotics, anti-virals, anti-fungals,anti-inflammatories, anticoagulants, lymphokines, and antigenicmaterials.
 120. The biologically active agent of claim 105, wherein saidfirst reaction center comprises an enzyme that is member of a classselected from the group consisting of oxidoreductases; transferases;hydrolases; isomerases; and ligases.
 121. The biologically active agentof claim 111, wherein said first reaction center comprises an enzyme inwhich said subject is deficient.
 122. A tissue assist device,comprising: a. a inorganic-based sol-gel matrix that is biocompatible;and, b. a first reaction center encapsulated in said matrix, whereinupon placing said biocompatible matrix in contact with fluids of asubject, said first reaction center converts a first prodrug into afirst biologically active agent, and wherein said first reaction centerprovides a biological function characteristic of tissue of said subject.123. The device of claim 122, wherein said biocompatible matrixcomprises a silica-based sol-gel matrix.
 124. The device of claim 123,wherein said tissue is an organ.
 125. The device of claim 124, whereinsaid organ is a liver.
 126. The device of claim 125, wherein said firstreaction center is one of the following: cytochrome P-450, hepatocytesor Kupffer cells.
 127. The device of claim 124, wherein said firstprodrug is endogenous to said subject and is more deleterious to saidsubject than said first biologically active agent.
 128. The device ofclaim 123, wherein said fluid is blood of said subject.
 129. The deviceof claim 125, wherein said first reaction center is xenogeneic.
 130. Thedevice of claim 123, wherein said contact occurs extracorporeal to saidsubject.
 131. The device of claim 123, wherein said tissue of saidsubject is deficient in converting said first prodrug into said firstbiologically active agent.
 132. A kit for treatment of a subject,comprising: a. a inorganic-based sol-gel matrix that is biocompatible;and, b. a first cell-free reaction center encapsulated in said matrix,wherein said first reaction center, after administration of said matrixto a subject, produces a therapeutically effective amount of a firstbiologically active agent from a first prodrug in said subject.
 133. Thekit of claim 132, wherein said biocompatible matrix comprises asilica-based sol-gel matrix.
 134. The kit of claim 133, furthercomprising instructions for treatment of said subject using said kit.135. The kit of claim 133, further comprising one or more doses of saidfirst prodrug for administration to said subject.
 136. The kit of claim135, wherein said dose of said first prodrug is formulated forcontrolled release of said first prodrug upon administration to saidsubject.
 137. A method of treatment of a subject, comprising: a. a stepfor encapsulating a first cell-free reaction center in a biocompatiblematrix; and b. a step for administering said biocompatible matrix to asubject; wherein said biocompatible matrix comprises a silica-basedsol-gel matrix, and wherein said first reaction center converts a firstprodrug into a first biologically active agent in said subject.
 138. Themethod of treatment of claim 137, further comprising a step foradministering said prodrug to said subject before, at the same time orafter said step for administering said biocompatible matrix to saidsubject.
 140. The medical article of claim 139, wherein said firstprodrug is endogenous to said subject and is more deleterious to saidsubject than said first biologically active agent.
 141. The medicalarticle of claim 139, wherein said fluid is blood of said subject. 142.The medical article of claim 139, wherein said article consists entirelyof said biocompatible matrix.
 143. The medical article of claim 140,wherein said article is implantable.
 144. The medical article of claim142, wherein said biocompatible matrix is attached to said article. 145.The medical article of claim 142, wherein said biocompatible matrix isattached to said article as a thin film.
 146. The medical article ofclaim 142, wherein said biocompatible matrix is attached to said articlein a capsule.
 147. The medical article of claim 139, wherein saidbiocompatible matrix is incorporated within said article.
 148. Themedical article of claim 139, wherein said article is a tissue assistdevice, wherein said first reaction center provides a biologicalfunction characteristic of tissue of said subject.
 149. The medicalarticle of claim 148, wherein said contact occurs extracorporeal to saidsubject.
 150. The medical article of claim 148, wherein said tissue ofsaid subject is deficient in converting said first prodrug into saidfirst biologically active agent.
 151. A method for producing a medicalarticle of claim 139 comprising: a. encapsulating a first cell-freereaction center in a biocompatible matrix; and b. shaping said matrixinto a desired morphology; wherein said biocompatible matrix comprisesan inorganic-based sol-gel matrix and wherein said first reaction centerconverts a first prodrug into a first biologically active agent. 152.The method of claim 151, wherein said matrix is cast into a morphologyselected from one of the following: cylindrical, rectangular,disk-shaped, patch-shaped, ovoid, stellate, or spherical.
 153. Themethod of claim 151, wherein said matrix is cast or sprayed as a thinfilm onto said medical article.
 154. The method of claim 151, whereinsaid biocompatible matrix comprises a silica-based sol-gel matrix. 155.The method of claim 151, wherein said biocompatible matrix is preparedfrom at least one type of oxysilane.
 156. The method of claim 155,wherein said biocompatible matrix is prepared from more than one type ofoxysilane.
 157. The method of claim 151, wherein said biocompatiblematrix is prepared from at least one type of inorganic oxide and atleast one type of oxysilane.
 158. A method for producing a medicalarticle of claim 139 comprising: a. encapsulating a first cell-freereaction center in a biocompatible matrix; b. crushing saidbiocompatible matrix; and c. encapsulating said crushed biocompatiblematrix; wherein said biocompatible matrix comprises an inorganic-basedsol-gel matrix and wherein said first reaction center converts a firstprodrug into a first biologically active agent.